Gastroenterology
Volume 133, Issue 1 , Pages 340-342, July 2007

Irritable Bowel Syndrome: Shifting the Focus Toward the Gut Microbiota

  • Fergus Shanahan

      Affiliations

    • Corresponding Author InformationAddress requests for reprints to: Fergus Shanahan, MD, Professor and Chair, Department of Medicine and Director, Alimentary Pharmabiotic Centre, University College Cork, National University of Ireland, Cork, Ireland. fax (353) 21-4345300.

Article Outline

 

See “The fecal microbiota of irritable bowel syndrome patients differs significantly from that of healthy subjects” by Kassinen A, Krogius-Kurikka L, Mäkivuokko H, Rinttilä T, Paulin L, Corander J, Malinen E, Apajalahti J, and Palva A, on page 24.

Microbes run the world.… Microbes are essential for every part of human life—indeed, all life on Earth.1

A leap in the understanding of chronic human disorders frequently requires a serendipitous discovery, a lateral shift in thinking, or a parallel advance in technology that enables hitherto impenetrable problems to be probed and hypotheses to be tested. It seems that all of these may be needed for the secrets of irritable bowel syndrome (IBS) to fall. The most common condition encountered by gastroenterologists and one of the more common in primary care, remains ill defined. A cause of suffering for many and disablement for some, IBS relies on symptoms for its diagnosis and largely on symptomatic remedies for treatment. Much of the difficulty is due to a lack of objective, reproducible biomarkers for defining the phenotype and subsets of the disorder. Therefore, reports from different research groups pointing toward new disease metrics or markers in IBS are welcome. These include evidence for immune activation, changes in cytokine levels, low-grade intestinal inflammation, and an altered stress response, particularly in patients without comorbidity, as measured by enhanced responsiveness of the hypothalamic–pituitary–adrenal axis.2, 3, 4

In addition to disturbances in the host response and the brain–gut axis, several unrelated observations have directed attention toward the gastrointestinal microbiota in patients with IBS. These include the existence of a postinfectious variant of the syndrome,5 epidemiologic evidence that antibiotic use may be a risk factor and linked with relapses,6 the proposal that some patients may have small bowel bacterial overgrowth,7 and reports of beneficial effects with specific antibiotics or probiotics.8, 9 It has been established from comparative studies of germ-free and colonized animals that the microbiota influence the structure and function of the gastrointestinal tract. Indeed, continual signaling from the enteric bacteria is required for mucosal homeostasis with regulatory effects, not only on the epithelium and immune system, but also on the enteric neuromuscular apparatus.10

The human gastrointestinal tract has the highest microbial cell density of any ecosystem on the planet. This living inner biomass has been estimated to contain up to 1014 cells, with 1012 organisms per gram of fecal material in the large intestine, and accounts for >1000 bacterial species. The microbial gene content (microbiome) exceeds that of the human genome, and collectively the metabolic capacity of the microbiota is tantamount to that of a hidden inner organ.10 Long neglected and considered difficult to study, several developments have renewed interest in the normal gut microbiota in health and disease. In particular, the limitations of traditional culture-based methods for studying bacteria have been circumvented with the advent of molecular techniques. Although most of the indigenous bacteria cannot be isolated in pure culture owing to difficulties simulating in vitro their niche within the enteric microenvironment, they can be surveyed molecularly.

Extraction of microbial nucleic acid from fecal or mucosal biopsy samples can be used to provide a profile of the composition of the resident microbes based on analysis of the small ribosomal subunit RNA gene. The small ribosomal subunit RNA (16S rRNA in the case of bacteria) contains highly conserved regions of base sequences that are interspersed with hypervariable regions that reflect evolutionary divergence of different bacterial species. The size of the 16S rRNA gene (about 1500 bp) is suitable for comparative sequence analysis and, by reference to large gene databanks, the isolate may be identified.11 Therefore, the sequencing of 16S rRNA genes of clones derived from fecal nucleic acid following amplification by polymerase chain reaction (PCR), serves as a method for profiling and phylogenetic classification of both culturable and nonculturable enteric bacteria. Molecular surveys based on 16S rRNA have suggested that the composition of the gut microbiota is stable but individual, and have revealed a remarkable diversity of organisms, including previously undescribed phylotypes. The strategy has also been used to detect reduced complexity in fecal microbial composition in Crohn’s disease.12

Molecular profiling of the microbiota has several pitfalls that may arise at the level of bacterial nucleic acid extraction, during PCR, or at the cloning steps. There is a selection bias in favor of the dominant groups of bacteria. This means that a difference between complex bacterial communities (such as in different fecal samples) might be missed if a small subgroup of bacteria is altered. To counter this problem, the introduction of an initial step, whereby the bacterial DNA is fractionated according to percentage guanine and cytosine (GC) content, before PCR, enriches the diversity of sequences obtained by cloning, and allows the less abundant species to be examined. This simple strategy presents the bacterial community structure as a percentage of total DNA versus percentage GC content of DNA and provides a representation of the relative abundance of phylogenetic groups of bacteria.13

In this issue of Gastroenterology, Kassinen et al14 report on the molecular profile of the fecal microbiota of patients with IBS, compared with that of control subjects. They have used the profile of GC content to remove the bias favoring the most abundant organisms and to select differences between IBS subtypes and controls for further analysis. The bacterial DNA preparations extracted from fecal samples were pooled; 10 from diarrhea-predominant (IBS-D) subjects, 8 from constipation-predominant (IBS-C), 6 from mixed type with alternating symptoms (IBS-M), and 23 controls. The pooled DNA preparations were then fractionated according to their percent GC content, and those fractions displaying the most divergence were selected for cloning and sequencing of the 16S rRNA gene. The results showed that in addition to differences at the level of GC fractional profiling between controls and patients and among the subsets of patients, there were also differences between the clone libraries for several bacterial genera, some of which seemed to be verified by quantitative PCR. The apparent alterations involved several bacterial genera with Lactobacillus sequences seeming to be absent from IBS and Collinsella sequences greatly reduced in IBS. The investigators previously studied the same patients and controls using traditional techniques with contradictory results, and propose that the present methods have more resolving power for showing diversity and alterations within the gut microbiota.

The results do not prove that the microbiota is abnormal in IBS. Rather, the overarching conclusion is that the microbial community is significantly altered in IBS and that the composition varies with the main symptoms. Whether such changes are causal, consequential, or merely the result of constipation and diarrhea is unclear. Clinical controls for these symptoms in the absence of IBS would have helped the interpretation of the results. Furthermore, the stability of the changes in the microbiota needs to be demonstrated, particularly for the so-called subsets of IBS (IBS-D, -C, and -M), because these may undergo transition over time.15

There are additional caveats to the interpretation of the results. First, the bacterial DNA samples were pooled, seemingly to enhance the opportunity to detect disease-related differences by overcoming individual variations, but this raises a doubt that GC fractionation of DNA samples grouped and pooled arbitrarily according to some other characteristic, such as age or gender, might exhibit similar differences. Second, the information obtained by sequencing of cloned 16S rDNA is not quantitative and the proportional composition of the total microbiota exhibiting differences from controls is unclear. This uncertainty regarding the extent and biologic significance of the observed differences in microbiota may have been compounded by the use of GC profiling to filter the areas for sequencing. Third, even allowing for the selection of genome fractions with divergences in GC content, the number of clones generated for sequencing in the present study was comparatively low (<4000 sequences from 47 subjects); whereas in metagenomic analyses of the human distal gut microbiome, Eckburg et al16 studied about 13,000 sequences from 3 individuals, and Gill et al17 about 1000 sequences for each of 2 subjects.17 In the future, the limitations and logistical constraints of molecular studies of the gut microbiota will be tackled by more ambitious metagenomic analyses with deeper coverage. These will probably require the collaborative input from research consortia, which are already underway on both sides of the Atlantic, and will exploit advances in high-throughput sequencing technology.1, 18

The solution to a chronic disease does not always reside solely within the host. Some gastrointestinal disorders will never be solved without due consideration of the residents within the lumen of the gut. This was the lesson of Helicobacter pylori and peptic ulcer disease. Could the same be true of IBS? The provocative report by Kassinen et al tells us more about how little we know than how much we have learned about the gut microbiota in health and disease. Reconciling alterations in the gut microbiota with other disparate disturbances in IBS is probably simplistic at this stage (Figure 1), but it will challenge investigators to generate and test hypotheses and enrich the scope of research in what is becoming an exciting area of endeavor.

  • View full-size image.
  • Figure 1. 

    Reconciling the disparate disturbances in IBS. Proinflammatory cytokine release due to low-grade intestinal inflammation may alter motor and secretory function and disturb the enteric microbial niche. The HPA axis is activated in response to centrally acting stressors or from peripheral cytokines and provides a link between the brain and immune function. Several factors may lead to disturbances in mucosal barrier function including inflammation and stress. AVP, argentine vasopressin; CRF, corticotrophin-releasing factor.

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References 

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PII: S0016-5085(07)01012-8

doi:10.1053/j.gastro.2007.05.030

Refers to article:

  • The Fecal Microbiota of Irritable Bowel Syndrome Patients Differs Significantly From That of Healthy Subjects , 17 April 2007

    Anna Kassinen, Lotta Krogius-Kurikka, Harri Mäkivuokko, Teemu Rinttilä, Lars Paulin, Jukka Corander, Erja Malinen, Juha Apajalahti, Airi Palva
    Gastroenterology July 2007 (Vol. 133, Issue 1, Pages 24-33)

Gastroenterology
Volume 133, Issue 1 , Pages 340-342, July 2007