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- Materials and methods
Irritable bowel syndrome (IBS) is a frequent functional gastrointestinal (GI) disorder defined by abdominal pain or discomfort and modifications in bowel habits in the absence of organic cause. Although morbidity of IBS remains very low, its detrimental impact on quality of life together with its worldwide prevalence (10–20%) and the absence of curative therapy explains the considerable economic impact of this disorder. The symptoms of IBS vary between affected individuals, but are better defined from a clinical point of view by the Rome criteria.[1, 3] The aetiology and pathophysiology of IBS remains poorly understood and is most likely multifactorial. Multiple interacting mechanisms may contribute to the development of IBS symptoms. Dysregulation of brain-gut interactions, generating gut dysmotility and visceral hypersensitivity, are considered as important factors in the pathology, although the causes of these features have not yet been determined. Other factors include psychological stress, low-grade inflammation potentially following GI infections and alteration within the gut microbiota.[5, 6]
The microbiota of the normal human intestine represents a complex mostly anaerobic ecosystem that plays a key role in maintenance of health and physiological functions of the host. This microbiota acts as a barrier against pathogens, stimulates the host immune system and produces a great variety of compounds from the metabolism of dietary and endogenous substrates that could affect the host. Disruption of the microbial ecosystem has been reported in different pathologies including inflammatory bowel disease and type-2 diabetes.[7, 8] Such microbial alteration may also be involved in the onset and maintenance of IBS. Indeed, IBS frequently follows antibiotic therapies or gastroenteritis. Furthermore, disturbances in the composition and stability of the gut microbiota were reported in IBS individuals compared with healthy ones.[9-14] Using standard cultural methods and culture-independent approaches, these studies showed abnormal variations within the faecal IBS microbiota affecting different bacterial groups, the most reproducible results concerning alterations in the Bifidobacterium and Clostridium coccoides – E. rectale subgroup. Specific IBS-related groups of microbes were not revealed from these studies. However, these approaches that quantified phylogenetic groups of bacteria could not assess the functional groups of microbes, i.e. all the bacterial species sharing the same metabolic activity.
Carbohydrate metabolism by gut microorganisms is a central process allowing supply of nutrients and energy to the host. This fermentative process is complex and involves several functional groups of bacteria with complementary metabolic activities that interact to ensure the biotransformation of polymers (resistant starch, nonstarch polysaccharides, proteins, mucins…) into end-products (mainly short chain fatty acids and gases). Hydrolytic communities transform complex substrates into smaller fragments that can also be used by other bacterial groups unable to hydrolyse polymers. Other microbial cross-feeding interactions are related to the utilisation of fermentative products such as succinate, lactate[15, 16] or hydrogen and involve specific groups of microorganisms. Elimination of hydrogen, the main gas produced from organic matter fermentation, is essential to maintain efficient fermentation in the gut. Its main route is utilisation by H2-consuming micro-organisms which comprise methanogenic archaea, sulphate-reducing and/or reductive acetogenic bacteria. Abnormality in microbial fermentation has already been suggested in IBS patients.[19, 20] In keeping with this, a range of fermentable dietary carbohydrates can exacerbate or provoke gastrointestinal symptoms through their fermentation by the gut microbiota.
We hypothesised that a functional dysbiosis might exist within IBS intestinal microbiota, inducing alteration in carbohydrate metabolism. We thus used a function-based approach of the ecosystem to compare the gut microbiota of IBS patients to that of healthy controls. This approach, which has been validated in healthy subjects, combines cultural evaluation of functional groups of microbes and fluorescent in situ hybridisation (FISH). Functional groups of microbes can only be quantified using specific cultural methods. By contrast, most of the phylogenetic groups composing the gut microbiota cannot be selectively cultivated and are quantified using molecular approaches based on 16S rDNA gene sequence. The metabolic capability of the IBS and healthy microbiota was further evaluated in vitro. Our work was focused on one IBS subtype, the constipated-IBS (C-IBS), to reduce heterogeneity between the IBS subjects studied, especially the variation in gut microbial composition due to different modifications of transit time. Only women were recruited for this study as they are more affected by IBS than men.
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- Materials and methods
Using a function-based approach to analyse the intestinal microbial ecosystem, we have demonstrated, in the present study, a critical functional dysbiosis in C-IBS gut microbiota, which can ultimately alter intestinal fermentative processes and host physiology. Microbial alterations identified here in C-IBS may be involved in genesis of different IBS symptoms, suggesting that these findings might be applicable to other IBS subtypes.
Previous studies have suggested that abnormalities of the intestinal microbiota occur in IBS.[9-14, 34, 35] However, pronounced deviations within taxonomic groups of bacteria were not identified. As previously reported, we found no difference between healthy and C-IBS subjects in either the total bacterial number or the major bacterial groups (Bacteroides, Lachnospiraceae and Ruminococcaceae) that compose the gut microbiota. A decrease in the lactic acid bacteria population (bifidobacteria and to a lesser extent, lactobacilli) was observed in faecal microbiota of our C-IBS patients, as also reported in several studies.[9, 10, 13, 14, 34, 35] The number of Enterobacteriaceae was shown to be increased in C-IBS compared with healthy individuals also as previously shown. Bifidobacteria are considered beneficial for the host in particular as they can inhibit growth of potential pathogenic bacteria. In C-IBS, the decrease in the bifidobacteria population may thus potentially affect gut heath by promoting growth of Enterobacteriaceae.
The function-based approach used in our study, has allowed demonstrating that the C-IBS gut microbiota is characterised by an important functional imbalance that was not detected using molecular approaches. Although molecular approaches mostly target one specific bacterial gene (16S ribosomal DNA gene), the functional approach is based on the detection of specific metabolic activity expressed by a group of bacterial species. This cultural method allows detecting and enumerating all viable microorganisms present in the gut, whatever their population level, whereas molecular approaches detected both dead and alive microbes with a detection limit closed to 106 to 107/g faeces for most of the methods.
Using the function-based approach, we did not observe significant differences in the distribution of predominant hydrolytic microorganisms involved in degradation of macromolecules such as fibre, protein or mucin, and belonging to the main bacterial groups of the gut microbiota, between C-IBS patients and controls. By contrast, we were able to identify important alterations in the population levels of major microbial groups involved in lactate and H2 metabolism as well as in butyrate synthesis. In particular, the C-IBS microbiota was characterised by a high number of lactate- and H2-utilising sulphate-reducing bacteria (SRB) compared with healthy subjects. Lactate and H2 are two of the main intermediate metabolites in the gut that support growth of various lactate-utilising and H2-consuming microorganisms. Among these microbial communities, SRB represent a group of bacteria that is able to use sulphate as terminal electron acceptor to form H2S with a wide range of substrates as electron donors, including lactate and H2. SRB are known to compete efficiently for utilisation of these two substrates in the human gut.[36, 37]
Lactate is quickly metabolised by specific bacterial species in the healthy gut microbiota into butyrate or propionate.[15, 22, 38] The number of these lactate-utilising bacteria was decreased 10-fold in the faecal microbiota of C-IBS patients compared with healthy ones. Concomitantly, the number of lactate-utilising SRB was increased by a 2 log-order in IBS compared with healthy subjects. This represents a major shift in the composition of the lactate-utilising community which is likely to be accompanied by a major shift in fermentation products. Lactate utilisation by SRB rather than by the non-SRB lactate-utilising community could explain the enhancement in sulphides production at the expense of butyrate formation observed in vitro in faecal sample incubations.
The slight decrease in butyrate production by C-IBS microbiota could further be due to the decrease in the number of certain butyrate-producing bacteria. FISH analysis showed that the population level of the Roseburia – E. rectale group (belonging to Lachnospiraceae), was lower in C-IBS subjects compared with control individuals, as previously reported. The reduction in butyrate production in C-IBS gut may reduce the potential health benefit from this metabolite, including anti-inflammatory effects, colonic defence barrier and decrease in oxidative stress. Butyrate oxidation by colonocytes was further shown to be altered by increasing H2S concentration.
Hydrogen is another important fermentative metabolite that is mostly removed from the ecosystem by H2-consuming microorganisms (methanogenic archaea, reductive acetogens or sulphate-reducing bacteria). In C-IBS, H2-utilising SRB were found in higher numbers than in healthy subjects, this increase coinciding with a decrease in the other H2-utilising microbial groups (i.e. acetogenic bacteria or methanogenic archaea). A shift in H2 metabolism may thus also exist in C-IBS subjects, contributing to increased sulphide production.
The predominance of the SRB population in the C-IBS gut microbiota should thus generate important shifts in fermentative pathways through alteration of inter-species transfers of lactate and H2. Results from in vitro fermentation of starch, one of the main polysaccharides available for gut microbes, further suggest that alterations in carbohydrate metabolism could exist in C-IBS gut microbiota, less butyrate and especially, more hydrogen and sulphide (H2S) being produced.
The functional dysbiosis observed in C-IBS microbiota may have important clinical implications, due to changes in metabolism output, and plays a major role in genesis and/or maintenance of different IBS symptoms including abdominal pain, modulation of gut transit and gas-related symptoms. In this context, the enhancement in SRB population and the consequent over-production of deleterious sulphides should have an important impact on IBS patho-physiology.
Abdominal pain is a prevalent symptom in IBS that is mainly related to enhancement in visceral sensitivity. More than 90% of IBS patients were shown to suffer from visceral hypersensitivity as measured by rectal distension. H2S was recently shown to have a major role in visceral nociception. Matsunami et al. reported that colonic luminal H2S could cause visceral pain-like nociceptive behaviour in mice through sensitisation/activation of T-type Ca2+ channels probably present in primary afferents. It is well known that colonic luminal H2S is mainly produced by SRB, with colonic tissues also forming some H2S from l-cysteine metabolism. Potential roles for colonic luminal H2S and/or SRB in inflammatory bowel diseases and colon cancer have been reported in several studies.[37, 43, 44] Our results support the hypothesis that H2S produced from SRB metabolism could play key role in human colonic pain.
H2S produced by SRB could also be involved in colonic transit regulation. Exogenous H2S was shown to inhibit in vitro motor patterns in human and rodent colon mainly through an action on multiple potassium channels. This is consistent with our finding suggesting higher H2S production in C-IBS subjects. Results from previous studies,  however, showed that the number of SRB was lower under conditions of slower gut transit in healthy volunteers, the transit time being, in this case, reduced artificially.
The stimulation of sulphate-reduction in C-IBS was also shown to alter H2 metabolism. This could further contribute to generate gas-related symptoms, i.e. bloating and flatus, which are frequently reported by IBS patients. An over-excretion of H2 in IBS patients was already reported by King et al. Similarly, in vitro starch fermentation by C-IBS faecal microbiota led to accumulation of H2 in the gas phase. Gas-related symptoms may thus be associated with H2 accumulation in the gut. Our results further suggest that this alteration could be due to a decreased capacity of the gut microbiota to re-utilise fermentative H2.
Some of the microbial changes observed in C-IBS may be consequences of the slower gut transit. In vitro continuous culture models have shown that dilution rate has an important impact on the composition of the human colonic microbial community.In vivo, a slower gut transit was shown to be related with higher methane-excretion in IBS. Similarly, the number of methane-producing subjects detected in our study was higher in C-IBS (8 over 14 subjects) than in healthy group (3 over 12 subjects). However, other important microbial alterations could not be explained simply by modification of gut transit and factors other than transit time may thus contribute to the altered microbial ecology observed in C-IBS. This suggests that certain of our findings on C-IBS gut microbiota might be applicable to other IBS subtypes.
In conclusion, we showed here, with a function-based approach, a major functional dysbiosis within gut microbiota of C-IBS. This cultural approach has allowed identifying variations within different functional groups of microorganisms that could have important physiological impacts for the host. This dysbiosis could indeed change the metabolic output and especially enhance production of toxic sulphides which could in turn influence motility and visceral sensitivity and generate IBS symptoms. The SRB community may thus have a central role in the microbial dysbiosis and in IBS patho-physiology. The contribution of SRB to IBS pathogenesis deserves further investigation and is currently under studies in our laboratory.