Our study is the first attempt at characterizing small bowel microbiota with massive parallel sequencing. We detected representatives of several genera with a predominance of Firmicutes. The prevalence of phyla in the jejunum was different from that in the stomach analysed by Andersson et al.21 using the same methodology. At the taxon level Streptococcus spp., Veillonella spp., Prevotella spp., Rothia spp., Haemophilus spp., Actinobacillus spp., Escherichia spp. and Fusobacterium spp. were the dominating species. Our results differ from those obtained by Zilberstain et al.27 in a study based on the direct aspiration and culture of jejunal fluid, describing domination of Veillonella spp., Lactobacillus spp., Proteus spp. and Bacteroides spp. in proximal jejunum. This difference is expected, since Zilberstain used culturing as the method of choice to characterize the microbiota and it is known that only a part of the bacterial colonizers of the gut can be cultured28,29. One previous study that used PCR-amplified 16S rDNA clone libraries to characterize the microbial diversity of a jejunum biopsy from a single healthy subject found 78% Firmicutes, 13% Proteobacteria, 3% Bacteroidetes, 1% Actinobacteria and 3% Fusobacteria30. Although these abundances were somewhat different from our mean values, they all fell within the ranges observed in our material.

454-Barcoded pyrosequencing is a powerful method to explore the diversity within the human gut ecosystems but it also has some limitations. One of them is the inability to distinguish between live and dead bacteria. Using jejunum biopsies instead of jejunal fluid we analysed mucosa-associated bacteria, thus diminishing the possibility of sequencing bacteria just passing the gastrointestinal tract. The mucosa-associated bacteria are of particular interest since they, generally speaking, are more likely to have a direct effect on the host through the mucosal layer than the bacteria only passing through the intestinal tract. We cannot exclude the contamination from oral or oesophageal flora although the probability is very low. We washed the Watson capsules before opening them and our method for DNA extraction was designed to extract only mucosa-associated flora. However, we found similarities at both phylum and taxon level with previously described microbiota from the distal oesophagus31. Franzosa et al.32 identified a subset of abundant oral microbes that routinely survive transit to the gut, but with a minimal transcriptional activity there. Although DNA from oral species was detectable in the gut it did not form a dominant component of that community.

We found no significant difference in small bowel microbiota between IBS patients and healthy controls. Our results do not correlate with some previous studies that reported differences between IBS patients and healthy controls in the composition of faecal microbiota14,33,34. Moreover, our findings do not support a role for SIBO in IBS. We did not find any qualitative differences in jejunum microbiota between patients and controls although we cannot exclude quantitative differences because the 16S amplicon cannot measure the absolute number of bacteria. The relationship between SIBO and IBS is highly inconsistent among studies35. SIBO is often diagnosed on the basis of various techniques for carbohydrate breath testing36. However, in a recent study Yu et al.37 demonstrated that lactulose breath testing detects oro-caecal transit, not small intestinal bacterial overgrowth in patients with IBS.

The achieved results do not reveal pronounced and reproducible IBS-related deviations of entire phylogenetic or functional microbial groups. The lack of apparent similarities in the taxonomy of microbiota in IBS patients may partially arise from the fact that the applied molecular methods, the nature and location of IBS subjects and the statistical power of previous studies have varied considerably15. It is unclear whether IBS is a disorder of the small intestine or the large intestine, or both. Our findings do not support a role for microbiota of the upper small intestine in the pathogenesis of IBS. However, we cannot rule out that changes in microbiota of the distal small bowel might influence development of IBS or IBS symptoms. Several studies have indicated that both epithelial barrier function and enteroendocrine function of the small intestine are important players in the crosstalk between intestinal microbes and the host that is believed to have an important role in the development of IBS38.

Regarding the degree of inter-individual variation of the gut microbiota in IBS, previous studies reported a highly significant loss of variation in IBS patients39 or suggested that the microbiota of IBS subjects was more heterogeneous than that of healthy controls40. We could not confirm such findings in our study.

Our study lends some support to the existence of a Prevotella enterotype in the small bowel but not the other two enterotypes described by Arumugam et al.5 This observation is in line with new data suggesting that the boundaries between the enterotypes may be fuzzier than previously suggested and the communities of gut bacteria may form a spectrum rather than falling into distinct groups41,42. The discrepancy can also be explained by different materials analysed in the two studies: faecal samples in the Arumugan study and small bowel mucosa samples in ours.

It is unclear whether or not Bacteroides versus Prevotella enterotypes exist as distinct entities or rather represent a continuum where the observed dietary associations occur at the extremes. Perhaps better described as an enterogradient between abundance of Bacteroides- and Prevotella- dominant gut microbial communities, it currently appears that these two genera do not coexist well within the gut environment. Organisms that are phylogenetically related and functionally similar tend to coexist within the same environment consistent with niche-driven community structures. Coexclusion of Bacteroides and Prevotella, taxonomically and functionally similar genera, within the gut is an exception perhaps suggesting competition within the same niche43. We cannot exclude that Prevotella enterotype reflects small bowel microbiota, as we found in the present study and Bacteroides large bowel microbiota. The abundance of Bacteroides versus Prevotella may be an oversimplification of alternative states of the gut microbiota in response to diet44.

Our study is the first to characterize small bowel microbiota with massive parallel sequencing. We did not confirm significant differences in small bowel mucosa-associated microbiota between patients with IBS and healthy individuals although we identified candidates for potentially being differentially expressed between controls and patients. Further studies are required to verify our results.