Utilization of culture-independent methods to analyze the microbial dynamics of ecosystems has revolutionized our view of the contribution bacteria make to their function and maintenance (1
). We used DGGE to generate 16S rRNA profiles of the gut microbiota and determined the temporal stability and diversity of members of several functionally important bacterial groups and how these groups varied while the Crohn's disease individual was in remission and relapse. One of the significant observations we report is that the temporal stability of species profiles from Crohn's disease remission and Crohn's disease relapse samples differed from those for the control samples. This is the first description of the instability of the bacterial community in Crohn's disease and highlights the need to take multiple samples from an individual when investigating their gut microbiota. We also found reduced community diversity with Crohn's disease, and these findings are in agreement with those of a previous study where 16S rRNA species profiles showed that the microbiota of Crohn's disease subjects is altered in active and quiescent disease (53
). The profiles from the Crohn's disease remission individuals were shown to be stable over a much longer time period than previously described in the literature. However, this stability is relative and was in no way comparable to that for the control group, which shows a much greater stability and a less dynamic bacterial community. A higher stability of the bacterial community for Crohn's disease remission patients may be one factor in maintaining remission, since in relapse a more variable community was observed, but due to the low numbers it was not considered significant and needs further analysis. We also concluded that there is a reduced level of stability and a change of diversity during relapse into an active disease state. The mean percent similarity of the DGGE profiles for Crohn's disease subjects at time zero was significantly lower than that for healthy subjects in this study, and this indicates greater host specificity.
How this stability can be maintained or even initiated needs further investigation, since it would be desirable to be able to show that remission and “a stable gut” were synonymous with each other. Since the bacterial community in healthy subjects is much more stable, it follows that this trait is desirable and is a feature of a healthy gut. If we were to speculate on the biological significance of this variation, one could envisage that changes in the bacterial composition over time would impact on the functions that this community is supplying to the host. Changes in bacterial functions, such as short-chain fatty acid production, not only will impact colonocytes (66
) but also can result in significant changes in numbers of other functionally important groups, such as the sulfate-reducing bacteria (4
), which in turn may trigger a response from the host. In addition, loss of butyrate producers, which have anti-inflammatory activity (50
), may result in the host suffering greater levels of inflammation in the gut. However, a much more in-depth analysis of the hosts' metabonome would need to be undertaken in order to verify which functions were changing in this community, and this variation would need to be correlated with changes in the bacterial groups. An alternative scenario is that variations in the gut microbiota result in the host's immune system responding too strongly to the changes and thus initiating inflammation. In a healthy gut where the community is relatively stable, the immune system is constantly sampling this collection of bacteria and each time regards it as “self” and does not initiate a robust inflammatory response. However, if the diversity of microbiota was fluctuating to a greater extent than one would find in a healthy subject, the immune system may “regard” the different numbers of bacteria as a significant threat, since they are outside the normal range of variation, and initiate a response which in turn results in damage to the host. One key element in this scenario is the trigger that causes an individual's gut microbiota to change from relatively stable to unstable. A potential candidate for the trigger may be genetic, e.g., a mutation in the CARD15/NOD2 locus, but there is evidence to suggest that the coincidence of this mutation with Crohn's disease and a reduced bacterial diversity is not 100% (44
). Furthermore, it is unclear which comes first, whether the change in stability of the gut microbiota triggers inflammation or vice versa.
Analysis of the C. leptum
subgroup, C. coccoides
subgroup, B. fragilis
spp., and LAB also revealed unexpected observations. The LAB profiles were complex, and temporal stability was low but still comparable to that of the control group. Nielsen and colleagues' analysis of the spatial distribution of LAB in biopsy samples revealed complex and variable communities with respect to sampling site and host (39
). Walter and colleagues (65
) previously observed the lack of stability in this bacterial group in healthy subjects and concluded that a significant proportion of the LAB populations were related to food-associated species. Indeed, the view of lactobacilli as allochthonous species in the intestinal tract has been recently proposed (60
), and this classification would account for their apparent instability in the gastrointestinal tract of all our samples. Previous culture-independent investigations of the LAB in Crohn's disease have shown differing results, with loss of diversity reported (44
) and no changes reported (38
). Thus, our observations add to the consensus of “loss of diversity” but temporally unstable and may suggest that diet needs to be more thoroughly controlled in these experiments in order to determine changes in the autochthonous LAB community.
A similar stable community was observed for the Bifidobacterium
spp. for all the groups investigated, and this observation has been previously reported (31
); however, this is the first report of stability of this group for Crohn's disease patients in remission and relapse. Culture-dependent approaches had shown that fewer Bifidobacterium
spp. were recovered from subjects with active Crohn's disease (13
); however, from our results we suggest that this observation is due to culturing bias and does not accurately reflect the dynamics of this group in Crohn's disease. We showed that the Bifidobacterium
population was stable for all three groups, and since they constitute approximately 5% of the total community (15
), we concluded that this group is not responsible for changes in the dominant community profiles of individuals. Furthermore, we would question their role in maintaining Crohn's disease in remission, since no significant changes were observed in the community structure during relapse.
The key result from the C. leptum
and B. fragilis
group-specific analysis was the significant failure to amplify the 16S rRNA gene products from members of the Crohn's disease group. These primers have been used extensively to amplify 16S rRNA gene products and have been found to very robust; therefore, any failure to amplify a signal with them was considered a significant result. While it is accepted that negative results are difficult to verify, we are confident that the results were genuine and were not due to inhibition of the PCR by factors coextracted with the DNA from the stool samples. All the DNA samples extracted here gave positive results for the universal V6-V8, LAB, and Bifidobacterium
primer sets. In the latter two cases, these bacteria are not present at the numbers we would expect for the two Clostridium
groups or the Bacteroides
group, which failed to be amplified for some subjects. If inhibition were the cause, we would expect it to be acting uniformly and not selectively on certain bacterial groups, and less abundant groups would also be affected. Hence, we strongly believe that these are valid observation. It is therefore conceivable that the Clostridium leptum
groups were present in a proportion of the initial samples at low numbers, which were below the threshold of detection for the specific primers used in this study. The inability to amplify C. leptum
group 16S rRNA gene products, we believe, is indicative of an underlying change in these groups in Crohn's disease individuals. Changes in numbers and diversity of the C. leptum
group in Crohn's disease have been previously found by using culture-independent approaches (53
) and more recently by using a metagenomic approach (19
We also observed that the Bacteroides fragilis
subgroup's diversity was significantly reduced in Crohn's disease, which included both remission and relapse, in addition to a failure to amplify this group from 39% of the samples. While other groups have not reported such dramatic changes in the Bacteroidetes
, we concluded that our observation was made possible due to the use of multiple samples from an individual rather than single-time-point samples. These two groups of bacteria are significant members of the gut ecosystem and play central roles in maintaining functions that are essential to gut health (2
). The phylum Bacteroidetes
has been shown to contribute to the host's ability to degrade indigestible carbohydrates (2
), while the members of the order Clostridiales
have been documented as being the main producers of short-chain fatty acids, such as butyrate, in the gut (8
). Thus, any changes in these keystone groups may impact on the total bacterial communities' capacity to provide beneficial functions to the host.
The role of the gastrointestinal microbiota in Crohn's disease is not fully understood, but the presence of a particular bacterial species or a component of the bacteria has been shown to be critical to onset of the disease (48
). This study indicates that the microbiota of Crohn's disease subjects is unstable over time compared to that of controls and that individuals with Crohn's disease have fewer bacterial species that are shared. It is not clear whether the apparent instability and atypical community present in certain Crohn's disease patients could reflect a dysregulation of host-specific immune responses to its commensals, whether this atypical community is a contributory factor to the disease, or if the atypical microbiota are present as a result of conditions in a diseased lumen that would favor their proliferation. However, one aspect which has emerged from this work and which warrants further investigation is the role of functional redundancy in the gut ecosystem. One characteristic which seems to be shared between individuals relates to the bacterial functions relevant to the gut, for example, butyrate production. While this function may be performed by different species or even genera in different individuals, the role seems to be sufficiently important in maintaining a healthy gut and is thus found in all individuals studied (46
). The common nature of these functions cannot be coincidence, since maintenance of these functions is important to the host; hence, any loss of these functions will affect not only the host but also the bacterial community. Several scenarios can be constructed which ultimately lead to situations detrimental to the host; for example, loss of short chain fatty acids synthesis results in an impact on the methanogen community and thus favors the growth of sulfate-reducing bacteria and the production of toxic hydrogen sulfite. The loss of butyrate producers may result in the loss of a potential anti-inflammatory agent (21
), which leads to more inflammation in the gut and a possible relapse; butyrate may affect inflammation by suppressing NFκB expression (51
). The observation reported here and elsewhere that the Clostridiales
communities are altered in Crohn's disease may indicate that we should shift our focus to understanding the functional roles bacteria play in maintaining a healthy gut and ask whether a loss of function, rather than specific organisms, plays a role in inflammatory bowel disease.