This study suggests that the faecal microbiota of patients with CD contains a markedly reduced diversity of Firmicutes. In particular, the Clostridium leptum
phylogenetic group was significantly less abundant in CD patients than in healthy subjects. This observation among patients in remission suggests that it could correspond to a primary modification (that is, a modification of the faecal microbiota that may exist before the onset of the disease and/or in the absence of major perturbations caused by the inflammation process). We focused on patients in quiescence because during the active phase of the disease the microbiota is remodelled6
and alterations in its composition could be a consequence more than a cause of inflammation.
A previous analysis of amplified and cloned rRNA gene libraries from healthy volunteers (n
3) and CD patients (n
also indicated that the Firmicutes (Clostridium leptum
and Clostridium coccoides
groups) had significantly lesser complexity in CD (25 v
73 OTUs) (Irène Mangin, personal communication). These results were obtained whether the subjects were considered individually or pooled. As in the present study, the number of ribotypes in the phylum Bacteroidetes was similar in the two libraries.
Although the cause of inflammatory bowel disease remains unknown, the indigenous intestinal microbiota is considered a major if not the main trigger of inflammation, both in animal models and in humans.24
A genetically determined abnormal response to indigenous bacteria is suspected, as well as dysbiosis.3,24
However, the precise microbial species or metabolites involved remain unknown. We share the current concept that the onset of CD could be due to an altered microbiota and that a dysbiosis could enhance the risk of disease. In that respect, the reduction in proportion of one microbial group is expected to be compensated for by a greater representation of others. In the present study, the less diverse and less represented microbial species in CD patients were Gram positive anaerobic bacteria that usually account for a major fraction of the faecal microbiota of healthy subjects. These species may provide CpG DNAs with immunomodulatory activities.25
A reduced proportion of Firmicutes may also be compensated for by an increased representation of Gram negative bacteria which are known to express more proinflammatory molecules such as lipopolysaccharide. In agreement with the latter point, we observed a specific association of Gram negative species of the Porphyromonadaceae family in CD patients.
In both of our libraries, the Firmicutes comprised mainly the Clostridium leptum
and Clostridium coccoides
groups, along with a number of unidentified species. These two groups have been described as essential components of the human indigenous intestinal microbiota.22,26
They contain all known microorganisms, producing large amounts of butyrate, which is not only the main energy source for colonic epithelial cells27
but also inhibits proinflammatory cytokine mRNA expression in the mucosa, by nuclear factor κB activation and IκB degradation.28
Loss of butyrate producers observed here could upset the dialogue between host epithelial cells and resident microorganisms, hence contributing to the development of CD associated ulcerations.
Our results emphasise the advantages of molecular methods over culture based approaches for comprehensive description of complex microbial communities.29
Indeed, we found 69% totally novel OTUs in CD patients, compared with 34% in healthy subjects, further supporting the existence of microbial dysbiosis in CD1,23
(figs 1, 2, respectively). Known OTUs identified in this study (corresponding to previously cultured microorganisms) corresponded to species known to reside in the human29
intestinal tract. These results are in good agreement with previous studies of human faeces using molecular approaches.23
Together, 16S rRNA sequence based studies of the human intestinal microbiota have led to the recognition of more than 50% totally novel species,31,32
suggesting that this community may be far more complex than previously thought.6
Our use of a DNA macroarray based strategy to analyse two large metagenomic libraries yielded evidence that CD may be characterised by a reduction in normal anaerobic bacterial diversity, especially among the Firmicutes, and did not support the role of a specific pathogen.
Although our study only involved a limited number of subjects (six healthy volunteers and six patients), our results were the same whether samples were pooled in the metagenomic approach or taken individually using the FISH method. Our observations point to formerly unrecognised specificities of the faecal microbiota of CD patients. Nevertheless, the number of subjects analysed was small as the metagenomic approach cannot be applied to a large set of samples. Our results should be confirmed by an epidemiological investigation focusing on the reduction in Firmicutes as well as over representation of species such as “uncultured Porphyromonadaceae”. Specific diagnostic tools could be designed to detect these. In addition, these would allow a more targeted use of antimicrobials or probiotics.
Genetic exploration of metagenomic libraries will provide access to the identification of genes and functionalities specific for CD. This is currently under investigation.