To our knowledge, this is one of the largest clone library studies investigating the microbiota in IBD. In contrast to an earlier study by Frank et al., , which examined a smaller number of clones from a large number of patients, we sought instead to add to current knowledge by obtaining a higher resolution of the IBD-associated microbiota with particular emphasis placed on observing differences between inflamed and non-inflamed colon sites in the same patients. This was inevitably done in a smaller number of patients and samples because of the depth of molecular analysis required for each sample. Our in-depth clone library analysis, utilizing the resolving power of near full-length 16S rRNA gene sequences, revealed significant differences in diversity and composition between the mucosal microbiota of healthy patients and IBD sufferers. The results also suggest a tendency towards a reduction in Firmicutes and an increase in Bacteroidetes species in IBD patients compared to controls and also indicate that there is an increase in Enterobacteriaceae in CD. Similar shifts in composition, in either one or all of these groups, have been reported by other investigators using both culture  and a variety of molecular techniques [29, 31, 45–55]. A previous large-scale clone library analysis by Frank et al., , however, reported a decrease in proportions of Bacteroidetes and the Firmicutes family Lachnospiraceae in a subset of, but not all, IBD patients and an increase in Proteobacteria. The observed discrepancies between these two large-scale clone library studies may in part be explained by different disease phenotypes, dietary or other environmental differences, the effect of inter-individual variation between patients or the differing number of samples studied and the depth of sequencing between each study.
We also demonstrated a reduction in bacterial diversity within IBD patients compared to controls and this is in agreement with several previous studies [24–27, 56, 57]. Our data shows, however, that despite the differences between IBD and non-IBD patients in both bacterial composition and diversity that samples clustered predominantly by individual rather than disease.
Using both culture and molecular methods, many studies have demonstrated that the mucosal community along the length of the colon is largely stable, in healthy and IBD patients, and distinct from that recovered in faeces [32–37]. Here we provide evidence instead for the development of localised differences in mucosal microbiota structure in IBD. Our community comparison results suggest that there may be differences between inflamed and non-inflamed tissue, with significant changes in the composition of the bacterial communities at these sites. A number of prior studies have also attempted to establish whether or not there is localised dysbiosis in IBD between inflamed and non-inflamed tissue. While two of these studies indicated that there is a dysbiosis [58, 59], the majority have suggested that this is not the case [29, 48, 60–62]. Discrepancies between these results and ours may result from the use of differing molecular methodology and/or the greater sequencing depth we employed. DGGE/TGGE and FISH are useful tools but the resolving power of these methods is much lower than that for in-depth clone libraries covering the full length of the 16S rRNA gene . In addition, DGGE/TGGE cannot accurately describe quantitative differences between dominant bands or describe qualitative differences in sub-dominant species and single bands on the gel may contain DNA from more than one species .
While our results suggest that localised changes in the mucosal microbiota do exist in IBD we were not able to identify a bacterial species or cluster that was consistently associated with the inflamed gut and therefore, potentially, with IBD aetiology. Other large-scale clone library analyses have also failed to identify specific pathogens [29, 30]. While their absence may indicate that potential pathogens may simply form a very minor component of the microbiota, these results do not support the hypothesis that a particular bacterial agent causes IBD. Clone libraries generate inherent biases, however, and it is possible that they are unable to detect certain species due to methodological artefacts. Indeed, this may be important with Mycobacterium avium subspecies paratuberculosis, a member of the often underrepresented Actinobacteria phylum [65, 66]. The absence of bifidobacteria from our dataset indicates that our clone libraries also suffer from this same bias against Actinobacteria. It is also worth noting that our analysis would not detect any viral, archaeal or eukaryotic aetiological agents. This may be important given recent evidence suggesting a role for viruses in the induction of at least some models of IBD .
Sequence-based microbiota comparisons such as ours can of course only demonstrate associations and do not provide information regarding mechanism or causation. It is also difficult to differentiate between compositional changes that may play a role in disease pathogenesis and those which may simply have occurred as a result of disease. However, given the absence of a specific and recurring aetiological agent in the cumulative data across all published IBD studies, which incorporate both culture- and molecular-based methodologies, it is possible that the alterations in bacterial composition and diversity seen between healthy and IBD patients and between inflamed and non-inflamed mucosa may be, to at least some extent, the result of the disturbed gut environment rather than the direct cause of disease. Indeed, there are a number of reasons why IBD is likely to result in altered conditions for bacterial growth. For example, the gut in IBD is likely to be a less stable environment than that of healthy individuals, with more exposure to antibiotics and other drug regimes, and alterations in transit time. Microscopy studies have suggested that there is a higher penetration of bacteria and a greater bacterial load in the mucosal layer in IBD patients [47, 68] and the resulting inflammation drives the localised release of antimicrobial compounds . In addition, in UC there is a reduced mucus layer in inflamed relative to non-inflamed regions .
Despite proportional increases in Enterobacteriaceae and Bacteroidetes within IBD patients, if these organisms were directly responsible for disease we might expect them to be elevated at sites of inflammation and this was not shown in our analysis. Taking into account all of the above factors, the observed increases in these bacterial groups in IBD patients as a whole may therefore simply reflect the adaptation of the individual microbiota to the IBD gut environment. Bacteroides thetaiotaomicron, for example, can adapt to inflammation in an experimental mouse model by inducing genes that metabolise host oxidative products  and inflammation per se has also been shown to promote the growth of Enterobacteriaceae in mouse models [72, 73]. Clearly, further similar studies are required on a far greater range of gut bacterial species so that we can better understand the response of the gut microbiota to alterations in environmental conditions.