Dysbiosis of the Intestinal Microbiota in IBS

The human gastrointestinal (GI) microbiota is a rich and dynamic community inhabited by approximately 1014 bacteria, most of which have not yet been cultivated in the laboratory (Zoetendal et al, 2006). The GI microbiota has been suggested as one of the etiological factors in irritable bowel syndrome (IBS), with a putative role in the development and maintenance of IBS symptoms (for a review, see Bolino & Bercik, 2010). The worldwide prevalence of IBS is 10-20% among adults and adolescents, depending on the diagnostic criteria applied (Longstreth et al, 2006). Abdominal pain or discomfort, irregular bowel movements and constipation or diarrhoea are common symptoms of IBS. Symptoms outside the GI tract, such as fatigue, anxiety and depression, are also often encountered. At its worst, IBS can cause significant effects on patients’ well-being, but it is not known to predispose to any severe illnesses. Patients can be grouped into three subtypes according to bowel habits: diarrhoea-predominant (IBS-D), constipation-predominant (IBS-C) or mixed-subtype (IBSM). However, the symptom subtype of each patient may vary over time (Longstreth et al, 2006). Compared to non-IBS controls, subjects with IBS have been associated with a greater temporal instability of the GI microbiota and quantitative changes have been detected within several distinct bacterial groups or species-like phylotypes, which are defined based solely on sequence data (see Table 1 for references). In analyses covering the overall microbial community, IBS subjects have shown a tendency to cluster apart from the healthy control subjects (Ponnusamy et al, 2011; Rajilić-Stojanović, 2007). Moreover, the IBS symptom-subgroups IBS have been proposed to differ from each other according to the GI microbiota of subjects within these groups (Lyra et al, 2009; Malinen et al, 2005; RajilicStojanovic, 2007). The most distinctive symptom sub-type is IBS-D, which could also be a result of the impact of the diarrhoea on the microbial environment in the gut. In addition, comparatively low quantities of bifidobacteria, which are generally considered beneficial to health, have been detected in several IBS studies (Balsari et al, 1982; Enck et al, 2009; Kerckhoffs et al, 2009; Krogius-Kurikka et al, 2009; Si et al, 2004). This finding, though still preliminary, encourages development of probiotic and prebiotic therapies for IBS. On the other hand, elevated numbers of Proteobacteria and Firmicutes, including Ruminococcus – like phylotypes, Lactobacillus sp. and Veillonella sp., have been reported. Quantitative and qualitative microbial alterations in the GI tract of IBS subjects may have a functional role in the syndrome aetiology or merely reflect the status of the gut, but still have diagnostic or prognostic value in clinical practise and research (Kassinen, 2009;


Introduction
The human gastrointestinal (GI) microbiota is a rich and dynamic community inhabited by approximately 10 14 bacteria, most of which have not yet been cultivated in the laboratory (Zoetendal et al, 2006).The GI microbiota has been suggested as one of the etiological factors in irritable bowel syndrome (IBS), with a putative role in the development and maintenance of IBS symptoms (for a review, see Bolino & Bercik, 2010).The worldwide prevalence of IBS is 10-20% among adults and adolescents, depending on the diagnostic criteria applied (Longstreth et al, 2006).Abdominal pain or discomfort, irregular bowel movements and constipation or diarrhoea are common symptoms of IBS.Symptoms outside the GI tract, such as fatigue, anxiety and depression, are also often encountered.At its worst, IBS can cause significant effects on patients' well-being, but it is not known to predispose to any severe illnesses.Patients can be grouped into three subtypes according to bowel habits: diarrhoea-predominant (IBS-D), constipation-predominant (IBS-C) or mixed-subtype (IBS-M).However, the symptom subtype of each patient may vary over time (Longstreth et al, 2006).Compared to non-IBS controls, subjects with IBS have been associated with a greater temporal instability of the GI microbiota and quantitative changes have been detected within several distinct bacterial groups or species-like phylotypes, which are defined based solely on sequence data (see Table 1 for references).In analyses covering the overall microbial community, IBS subjects have shown a tendency to cluster apart from the healthy control subjects (Ponnusamy et al, 2011;Rajilić-Stojanović, 2007).Moreover, the IBS symptom-subgroups IBS have been proposed to differ from each other according to the GI microbiota of subjects within these groups (Lyra et al, 2009;Malinen et al, 2005;Rajilic-Stojanovic, 2007).The most distinctive symptom sub-type is IBS-D, which could also be a result of the impact of the diarrhoea on the microbial environment in the gut.In addition, comparatively low quantities of bifidobacteria, which are generally considered beneficial to health, have been detected in several IBS studies (Balsari et al, 1982;Enck et al, 2009;Kerckhoffs et al, 2009;Krogius-Kurikka et al, 2009;Si et al, 2004).This finding, though still preliminary, encourages development of probiotic and prebiotic therapies for IBS.On the other hand, elevated numbers of Proteobacteria and Firmicutes, including Ruminococcuslike phylotypes, Lactobacillus sp. and Veillonella sp., have been reported.Quantitative and qualitative microbial alterations in the GI tract of IBS subjects may have a functional role in the syndrome aetiology or merely reflect the status of the gut, but still have diagnostic or prognostic value in clinical practise and research (Kassinen, 2009;Salonen et al, 2010).In the following chapter, these IBS-related alterations within the human GI microbiota are reviewed.

Intestinal microbiota
The intestinal microbiota is individual-specific and relatively stable through time (Zoetendal et al, 1998).From a microbial point of view, a tremendous variety of physiologically connected environments exists in the human GI tract.The mouth and stomach harbour their distinct microbiotas (Bik et al, 2006;Zaura et al, 2009).In the small intestine, the bacterial load and diversity rise from 10 4 to 10 8 cells per millilitre of intestinal content towards the distal ileum.Veillonella, Streptococcus, Clostridium cluster I and Enterococcus form the core genera of the small intestinal lumen (Booijink et al, 2010).Reaching the colon, the transit slows down and the bacterial density rises from 10 8 in the caecum and ascending colon to an average of 10 11 to 10 12 cells of bacteria per gram in faeces.The proportion of obligate anaerobic bacteria expands to 99%.The phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria and Verrucomicrobia are present in the colon (Andersson et al, 2008;Kurokawa et al, 2007).In the small and large intestines, the mucosal and luminal microbiotas are distinct from each other (Booijink et al, 2007;Zoetendal et al, 2002).Recently it has been shown that the human GI microbiota roughly groups into three entorotypes, with either Bacteroides, Prevotella or Ruminococcus predominating (Arumugam et al, 2011).The GI microbiota has a dynamic mutualistic relationship with its host affecting host nutrition and metabolism, immunocompetence and tolerance, GI tract surface maturation and function and even behaviour, thus possessing a potentially tremendous impact on host well-being (for review see Sekirov et al, 2010).Multiple theories linking IBS aetiology with the intestinal microbiota have been proposed, which, together with the discovered IBSassociated GI microbiota alterations, imply that bacteria may well play a role in IBS aetiology.

Gut microbiota in IBS
Alterations in the GI microbiota related to IBS have been investigated since the early 1980s' by conventional culture-based methods and an array of molecular methods (Table 1).Several of the published studies are based on the same Finnish sample panel originating from a probiotic intervention and additional healthy control subjects.For clarity, these studies are represented under a separate sub-heading in Table 1.Besides differing from control subjects, IBS also differs from IBD including Crohn's disease and ulcerative colitis (Enck et al, 2009;Ponnusamy et al, 2011).

Study
Samples Method Outcome for IBS 1 subjects Balsari et al, 1982 20  The sample set consisted of probiotic intervention samples from the intervnetion by Kajander et al., (2005) and additional control samples from subjects devoid of gastrointestinal symptoms.The detected alterations are given under the sample group they apply to. 3 The probiotic supplement was a combination of L. rhamnosus GG and Lc705, B. breve Bb99 and P. freudenreichii ssp.shermanii JS administered for 6 months at a daily dose of 8-9 x 10 9 CFU with equal amounts of each strain (Kajander et al, 2005).

Culture-based analyses
Using culture-based techniques, the GI microbiota of IBS patients was characterized as having less coliforms, lactobacilli and bifidobacteria in a study with 20 IBS patients and 20 controls (Balsari et al, 1982).Likewise, in a later study (Si et al, 2004) with 25 IBS patients fulfilling the Rome II criteria and 25 controls, lower levels of bifidobacteria were detected in IBS patients, but the level of bacteria belonging to the family Enterobacteriaceae was higher in IBS patients.Contrary to Balsari et al. (1982), Mättö et al. (2005) detected more coliforms in IBS subjects' and no difference in the bifidobacterial counts using culture-based methods, whereas the number of coliforms and aerobe:anaerobe ratio were elevated (26 IBS and 25 control subjects).In 2009, Enck and colleagues conducted an impressive culturing study by analysing the intestinal microbiota of a total of 34 313 subjects of varying conditions (Enck et al, 2009).Routine analyses were applied to Clostridium difficile, Bifidobacterium spp., Bacteroides spp., Escherichia coli, Enterococcus spp.and Lactobacillus spp.A total of 7 765 IBS subjects were included in the final data analysis revealing a significantly lower abundance of Bifidobaterium spp.In the latest study based on culturing, aerobes were elevated in the faecal samples of IBS-D patients compared with control subjects, whereas anaerobes, Clostridium spp., Bacteroides spp., Lactobacillus spp., Bifidobacterium spp., and Escherichia coli were not altered in IBS-D (Carroll et al, 2010).Taken together, evidence for increased numbers of aerobes and comparably low counts of bifidobacteria exist from culture based analyses with the latter giving good grounds for prebiotic and probiotic therapy research.The results on coliforms are contradictory between different studies.

Community structure with molecular methods
The overall microbial community from faecal samples of IBS subjects has been analysed applying denaturing gradient gel electrophoresis (DGGE), microarray (HITCip and PhyloChip), and sequencing (conventional Sanger sequencing and 2nd generation 454 pyrosequencing).All of these methods are capable of detecting the unculturable species in the microbiota, although they bear restrictions due to primer and probe dependency and technical biases.The main advantage is the possibility to gain a non-restricted overview and with sequencing, to able to design targeted primers and probes for applications based on PCR or hybridization.
Greater temporal instability of the intestinal microbiota of IBS patients compared with that of healthy controls has been detected with RNA-based DGGE (Maukonen et al, 2006).
Applying DNA-based DGGE on the same sample set did not show IBS related temporal variation (Maukonen et al, 2006), but variation due to antibiotic therapy was observed (Mättö et al, 2005).The inter-individual variability has been assessed in two studies with contradictory results.With the HITChip microarray analysis IBS subjects showed significantly more inter-individual variation compared with the controls (Rajilić-Stojanović, 2007), whereas with DGGE more variation was seen amongst control subjects (Codling et al, 2010).This discrepancy is likely due to methodological differences as the probe or primer based bacterial targets differ.Moreover, the biodiversity, an expression of species richness and abundance, is decreased in IBS (Noor et al, 2010).Loss of species richness was especially evident among Bacteroides species, which was speculated to suggest their putative protective role in the GI tract (Noor et al, 2010).Krogius-Kurikka et al. (2009) also found IBS-D related GI microbiota to lack diversity in a sequencing analysis, but the number of samples pooled prior to analysis was lower in the IBS-D sample possibly affecting the result (Krogius-Kurikka et al, 2009).On the other hand, Saulnier and colleagues (2011) analysed multiple samples from 22 pediatric IBS patients and 22 control subjects with 454 pyrosequencing (54 287 reads per sample) and a portion of the samples further with the PhyloChip which targets revealing no significant difference in bacterial bacterial quantities or richness, although qualitative changes were detected between the two subject groups and in relation to perception of pain (Saulnier et al, 2011).Thus, at least for adult IBS subjects, the diversity and species richness in the GI microbiota are diminished, which would together with the alternating symptoms explain a higher temporal and inter-individual variation in the gut microbiota.A less stable microbiota is potentially more vulnerable to external interference (infection, antibiotics, stress), possibly leading to a recurrent aberration in gut function.

From community to phylotype level
In addition to the overall community structure and stability in the GI tract, the thousands of bacterial species therein, referred to as phylotypes when based only on molecular data, are important.Aspects such as their absolute and relative abundance, prevalence and association to symptoms sub-types and perception have been studied.(less abundant in IBS-D than in all other groups).The Clostridium coccoides and Bifidobacterium catenulatum group assays detected more target bacteria in controls than in IBS patients when the results from the three different time-points were averaged and the IBS subjects analysed as a single group.
Thereafter, the samples were analysed with percent guanine plus cytosine (%G+C) profiling (Kassinen et al, 2007): The pooled symptom subtype profiles diverged with the %G+C profiling and the three most diverging fractions were subsequently studied using 16S rDNA Sanger sequencing.Real-time PCR assays targeting specifically the alterations between the sequesnce libraries of IBS and control subjects were designed and applied in several studies highlighting a ruminococcal phylotype in relation to IBS-D and a taxonomically unclassifiable phylotype with the control subjects and IBS subjects under probiotic theraphy (Lyra et al, 2010;Lyra et al, 2009;Malinen et al, 2010).Ruminococcal bacteria have also been associated with Crohn's disease and pediatric IBS (Frank et al, 2007;Martinez-Medina et al, 2006;Saulnier et al, 2011).Ruminococci include mucolytic bacteria with a possible competitive advantage in a disturbed gut with excessive mucus secretion.Novel uncultured bacterial phylotypes discovered in relation to IBS and health may also perform well as diagnostic microbiome signatures (Kassinen et al, 2007;Lyra et al, 2009;Saulnier et al, 2011) although their possible relation to the syndrome is mere speculation at this stage.The genera Bifidobacterium, Coriobacterium and Collinsella within the phylum Actinobacteria have been less abundant in IBS patients (Enck et al, 2009;Kassinen et al, 2007;Kerckhoffs et al, 2009;Lyra et al, 2009).Correspondingly, redused levels of Actinobacteria including bifidobacteria have been associated with Crohn's disease patients www.intechopen.com(Fyderek et al, 2009;Manichanh et al, 2006;Sokol et al, 2009), and Collinsella aerofaciens has been associated with a low risk of colon cancer (Moore & Moore, 1995).These more specific changes, once well established in relation to both healthy and non-IBS GI patient controls, have potential in diagnostics and tailor-made therapeutic approaches.
The possibility of finding a causative agent for IBS, for instance among the ruminococcal phylotypes, is intriguing though still a future challenge.The other microarray analysis was done on pediatric IBS subjects applying the PhyloChip (Saulnier et al, 2011).PhyloChip targets a wider array of microbes not specifically restricted to the expected human intestinal tract inhabitants (Brodie et al, 2006) although it is well applicable also to analysing intestinal microbiota (Nelson et al, 2011;Saulnier et al, 2011).Saulnier and colleagues (2011) discovered that Proteobacteria, especially Gammaproteobacteria, are abundant in pediatric IBS subjects.Within these Gammaproteobacteria the species Haemophilus parainfluenzae was commonly encountered.Similarly the pyrosequencing analysis revealed elevated numbers of Protebacteria and unclassified ruminococcal phylotypes in association to pediatric IBS and (Saulnier et al, 2011).In the PhyloChip analysis, several Bacteroides phylotypes, including a Bacteroides vulgatus -like phylotype, were elevated in healthy children (Saulnier et al, 2011), as has previously been noted in the case of adult subjects with ulcerative colitis (UC) or IBS (Noor et al, 2010).These efficient high-throughput methods have potential for analyzing large enough sample sets to identify common alterations in the heterogeneous IBS subject population.So far too few studies have been published for making a consensus on the results.In addition, it would be beneficial if the sampling schema would include several samples linked to thorough symptom data from each subject as both the microbiota and the symptoms in IBS are prone to alter over time.

The mucosal microbiota
The mucosal microbiota is of special interest in health related studies as it is in an intimate contact with the host.In a healthy intestine, the mucosal microbiota resides on the mucosal lining of the epithelium, whereas in a damaged intestine straight contact with the host epithelium is plausible.Fluorescent in situ hybridization (FISH) applied on mucosal samples of patients with IBD, IBS or no GI symptoms revealed that mucosal bacteria were more abundant in IBS patients than in healthy controls, although the difference was less evident than with the IBD patients (Swidsinski et al, 2005).The proportional amounts of the different bacterial groups targeted in the FISH analysis (Bacteroides-Prevotella, Bacteroides fragilis, Eubacterium rectale-Clostridium coccoides, Faecalibacterium prausnitzii and Enterococcus faecalis), however, were similar between IBS patients and controls (Swidsinski et al, 2005).Likewise, Carroll and colleagues (2010) found no significant difference between the abundances of cultured bacteria (aerobic, anaerobes, Clostridium spp., Bacteroides spp., Lactobacillus spp., Bifidobacterium spp., and Escherichia coli).With qPCR, elevated levels of Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, have been detected in duodenal brush samples of IBS patients.Nevertheless, a recent terminal-restriction fragment length polymorphism (T-RFLP) analysis was able to differentiate between the composition of IBS-D patients' and control subjects' mucosal microbiota, although the overall microbial profiles clustered according to site of sampling (mucosal or luminal) rather than health status (IBS-D or healthy control) (Carroll et al, 2011).Taken together, no drastic alteration in the mucosal microbiota of IBS subjects has been defined.The mucosal and luminal microbiotas differ in IBS subjects as they do in healthy controls, underlying the importance of research on this specific niche.One reason for the small number of mucosal IBS studies is the invasive nature of mucosal sampling as colonoscopy is not a regular procedure in IBS diagnostics or treatment.

Short Chain Fatty Acids (SCFAs)
The principal products of microbial carbohydrate metabolism in the human GI tract are short-chain fatty acids (SCFAs), which can be absorbed by the human host.The SCFAs produced throughout the GI tract are mainly acetate, butyrate and propionate, but in the colon acetate predominates (Cummings et al, 1987).The colonic epithelial cells prefers butyrate over other SCFAs as an energy source, and butyrate has been shown to have a positive effect on health (Pryde et al, 2002).The most abundant intestinal butyrateproducing bacteria are Firmicutes from Clostridial clusters XIVa and IV (Clostridium, Eubacterium, Fusobacterium) (Pryde et al, 2002).Starch fermentation by starch-degrading bacteria results in comparatively high amounts of butyrate (Chassard et al, 2008).Starchdegrading bacteria, including Ruminococcus bromii (Clostridium cluster IV), Eubacterium rectale (Clostridium cluster XIVa) and bifidobacteria (Leitch et al, 2007), comprise approximately 10% of culturable bacteria in faecal samples (Chassard et al, 2008).Reduced amounts of total SCFAs due to lower levels of acetate and propionate have been measured in association with IBS-D, while an elevated concentration of n-butyrate seemed to be characteristic of IBS-D (Treem et al, 1996).Tana and colleagues (2010) analysed the microbiota and SCFAs from faecal samples donated by 26 IBS and 26 control subjects.Contrary to Treem et al. (1996), the IBS subjects had elevated numbers of Veillonella spp.and Lactobacillus spp.together with higher concentrations of total organic acids and acetic and propionic acid.The increase in acidic metabolites was more pronounced in the group of IBS subjects with worse GI symptoms, quality of life and emotional status according to subjective evaluation (Tana et al, 2010).Colonic gas production (H 2 and CH 4 ) has been shown to be greater in patients with IBS (Rome II criteria) compared with controls using a standardized diet, which might be associated with alterations in the activity of hydrogen-consuming bacteria (King et al, 1998).An exclusion diet, mainly devoid of dairy products and cereals other than rice, reduced IBS symptoms and lowered the maximum gas excretion (King et al, 1998).Furthermore, functional constipation and IBS-C have been associated with methane production according to breath testing in a recent meta-analysis (Kunkel et al, 2011).Thus, although the results on microbial metabolites in the colon are still scarce and to some extent contradictory, they have been linked to symptom severity and defecation habit subtype.The elevated amount of butyrate among IBS-D subjects in one study is surprising, as butyrate is considered to have a positive effect on health.

Luminal proteases
Certain studies have suggested an association between luminal proteases and IBS.An elevated faecal serine protease activity has been associated with IBS-D (Roka et al, 2007).The faecal supernatants from IBS-D patients caused increased colonic paracellular permeability when administered to the mucosal side of a mouse colon strip and increased visceral hypersensitivity in mice (Gecse et al, 2008).Gecse et al. (2008) also showed that the effect on mucosal permeability is mediated by serine protease through protease-activated receptor two (PAR-2).Pre-incubation with serine protease inhibitors decreased the effect of the faecal supernatant from the IBS-D patients on the colonic paracellular permeability of mouse colon strips.Furthermore, the use of colonic strips derived from PAR-2-deficient mice completely removed the increase in colonic paracellular permeability.The elevated serine protease activity in IBS-D patients was suggested to be of microbial origin (Gecse et al, 2008).The PAR-2 mediated increase in visceral hypersensitivity appears to be specifically related to IBS-D in comparison to inflammatory bowel diseases (IBD) (Annahazi et al, 2009).The evidence for the role of luminal proteases in IBS symptoms is at its early stage, but intriguing.It links the GI microbiota with the host's IBS symptoms through increased gut permeability and visceral hypersensitivity.

Post-infectious IBS
In a large cohort study (over 500 000 patients), gastroenteritis was concluded to increase the risk of developing IBS by a factor of ten (Rodriguez & Ruigomez, 1999).Post-infectious IBS (PI-IBS) has been reported after Campylobacter, Shigella and Salmonella infections (Ji et al, 2005;Mearin et al, 2005;Spiller et al, 2000) and Staphylococcus aureus has been detected in a comparatively high prevalence in IBS subjects (Rinttilä et al, 2011).Nevertheless, PI-IBS appears to be a non-specific response (Spiller, 2007).Typically PI-IBS is characterized by loose stools, less depression and anxiety and increased enterochromaffin cells in mucosal biopsies compared with non-PI-IBS (Dunlop et al, 2003;Neal et al, 2002).Detecting an infectious agent from random IBS subjects is unlikely (Rinttilä et al, 2011), but this still does not rule out the possibility of an earlier infectious event having etiological importance.Since the initial gastroenteritis triggering PI-IBS is a coincidental event, and among PI-IBS patients the symptoms are relatively homogeneous and psychological abnormalities are less common than in other IBS patients, PI-IBS presents a clearer model for studying the possible mechanisms underlying IBS (Spiller, 2007).On the other hand, PI-IBS may be etiologically too distinct to represent the whole of IBS subtype variety.In addition to acute gastroenteritis triggering IBS symptomology, low-grade inflammation with focus on mast cells and monocytes has been suggested to have a pivotal role in IBS aetiology (for review see Ohman & Simren, 2010).Low-grade mucosal inflammation (Barbara et al, 2007;Chadwick et al, 2002;Dunlop et al, 2003;Ohman et al, 2005) and stable alterations in mucosal gene expression (Aerssens et al, 2008) of IBS patients of all symptom subtypes have been detected.Furthermore, the basal and E. coli lipopolysaccharide induced release of pro-inflammatory cytokines from peripheral blood mononuclear cells has been shown to be elevated in IBS-D patients compared to healthy controls (Liebregts et al, 2007).Additionally, antibodies against certain bacterial flagellin have been detected in IBS patients, particularly in PI-IBS patients, with a higher frequency than in healthy controls (Schoepfer et al, 2008).Taken together, PI-IBS is a widely accepted sub-type of IBS which can reside from a variety of causative agents.Minimizing risk, severity and length of acute gastroenteritis would likely lower the risk of recurrent functional GI disturbances such as IBS.

Probiotics for balancing the GI microbiota in IBS
Being a syndrome diagnosed based on subjective assessment of GI function, the most important outcome in IBS intervention studies is the patients' sensation of symptom relief.This is usually assessed by applying GI symptom questionnaires.According to a recent meta-analysis, the separate IBS symptoms (abdominal pain, bloating and flatulence) and their composite sum have all been significantly improved with probiotics (Hoveyda et al.

2009).
From the microbiological point of view, it is of interest to see whether the symptom improvement during the intervention is linked to alteration within the GI microbiotaputatively to a state that better resembles that of healthy-like control subjects.If no change in the microbiota is seen in a specific study, this doesn't necessarily mean there hasn't been one as the methodology used may have missed the targets of interest.This has been the case for a multispecies supplement intervention trial with L. rhamnosus GG, L. rhamnosus LC705, B. breve Bb99 and P. freudenreichii ssp.shermanii JS (Kajander et al, 2005), first assessed by qPCR assays targeting known GI bacteria (Kajander et al, 2007) and thereafter by targeting IBS associated phylotypes (Lyra et al, 2010).The analyses of 300 known GI bacteria showed no alterations due to the intervention (Kajander et al, 2007), but a vast number of phylotypes may have been missed with analyses restricted by primer target selection, whereas in the latter study, when the same samples were screened with qPCR assays targeting specifically IBS associated phylotypes (Lyra et al, 2010), alterations towards levels previously measured in controls devoid of GI symptoms (Lyra et al, 2009) were measured.Nobaek and co-workers (2000) have analysed abundances of Enterobacteriacea, sulphatereducing bacteria and Enterococci in IBS subjects consuming a rose-hip drink with Lactobacillus plantarum (DSM 9843).No alterations were detected in the probiotic group, but the probiotic strain was detected in faecal and rectal mucosal samples (Nobaek et al, 2000).Here again, the analysis method covered only a minor portion of the entire microbiota.In addition to affecting the bacterial levels and the stability of the GI microbiota, the relief of bloating and distention with probiotics may be linked to an effect on microbial metabolism (Schmulson & Chang, 2011).

Conclusion
Dysbiosis of the intestinal microbiota in IBS has been detected on several levels: the overall community appears to be less diverse with more variation between individuals and over time.These phenomena may reduce the resilience of the microbiota to external stressors, and both trigger and sustain functional aberrations in the gut.In addition to overall dysbiosis, specific bacterial groups are either elevated (Lactobacillus, Veillonella, Ruminococcus, Enterobacteria, aerobes as a group, S. aureus) or reduced (Bifidobactrium, B. catenulatum, Bacteroides) in IBS, but with the exception of bifidobacteria, the available data is not yet conclusive.Ruminococcal phylotypes have been associated specifically with IBS and also with inflammatory states in the intestinal tract in several studies and certainly deserve more attention.The analytical methodologies available for studying the GI microbiota have developed immensely in the past decade and the discovery of efficient microbial signature based diagnostic and therapeutic methods even for such a heterogeneous and subjectively defined patient group as IBS can be expected in the near future.
The sample set studied byMättö et al. (2005) andMaukonen et al. (2006) was further studied using 20 quantitative real-time PCR (qPCR) assays covering approximately 300 bacterial species (27 IBS patients and 22 controls gave faecal samples at the first time-point; 21 IBS patients and 15 controls gave faecal samples at three time-points at three-month intervals)(Malinen et al, 2005).The first time-point was analysed with IBS subjects divided into symptom subgroups; IBS-D, IBS-C and IBS-M.Statistically significant differences were observed with real-time PCR assays targeting Lactobacillus spp.(less abundant in IBS-D than in IBS-C), Veillonella spp.(less abundant in controls than in IBS-C) and Bifidobacterium spp.

Allistipes correlated with pain Studies on a Finnish sample set 2 Study / Method Healthy controls IBS subjects on placebo IBS subjects on probiotic 3
1The abbreviations in order of appearance stand for IBS, irritable bowel syndrome; HITCip, Human Intestinal Tract Chip; FISH, fluorescent in situ hybridization; qPCR, real-time quantitative PCR; CD, Crohn's disease; UC, ulcerative colitis; GI, gastrointestinal; DGGE, denaturing gradient gel electrophoresis; SCFA, short chain fatty acids; IBS-D, diarrhea-predominant IBS; T-RFLP, terminalrestriction fragment length polymorphism; NA, not analyzed; and IBS-C, constipation-predominant IBS. 2