Using a molecular approach, we observed that the faecal microflora in patients with both inactive and active colonic CD differed from the faecal microflora of healthy subjects, containing significantly more enterobacteria. In addition, approximately 30% of the dominant bacteria did not belong to the usual dominant phylogenetic groups. During active disease, the equilibrium of the dominant microflora was disturbed but species diversity remained high.
Several lines of evidence point to a role of the endogenous microflora in the pathogenesis of CD. Intestinal lesions predominate in the distal parts of the gastrointestinal tract where the intestinal microflora is most abundant, and bacteria present in the faecal stream have been reported to be responsible for the recurrence of intestinal lesions after surgery.3,4,24,25
Less than 30% of the endogenous colonic bacteria can be cultured.10,23
Modern molecular techniques based on nucleic acid sequence comparisons are powerful tools for culture independent characterisation of the microbial composition of complex ecosystems,26
and give a better representation of the dominant bacterial populations. The methods used in the present study allowed the detection of a phylogenetic group (dot blot) or a bacterial species (individual 16S rRNA sequence in TTGE) only if it represents at least 1% of the total bacterial microflora27
—that is, approximately 108
bacteria per gram. Such methods cannot detect bacteria of low concentration, some of which may be identified by culture techniques.
To examine primary alterations of the endogenous microbiota during remission, we selected CD patients with colonic disease who had not received antibiotics, sulphasalazine, or colonic cleansing for at least four weeks before analysis. Our patients received treatments required for the maintenance of remission—that is, mesalazine, corticosteroids, or immunosuppressors. The effects of such treatments on the composition of the microflora are unknown. We decided not to study patients treated with sulphasalazine as it may influence the microflora, probably because of the sulphonamide moiety.28
The increase in enterobacteria during both inactive and active CD has already been suggested in culture based studies.29,30 Escherichia coli
strains have been implicated in the pathogenesis of CD, especially some strains with particular adhesion properties which are increased in ileal lesions of CD.31
In contrast, however, E coli
strain Nissle 1917 may have a protective effect when administered orally, as suggested by clinical trials in patients with ulcerative colitis32–34
exhibited proinflammatory properties in several animal models of IBD.35
While some culture studies have suggested a possible increase in faecal Bacteroides
we observed a decrease in the relative proportions of the Bacteroides
phylogenetic group. The group probe hybridises with Bacteroides
but also members of the Porphyromonas
genera, and cannot detect subtle specific modifications of Bacteroides
at the species level, for example B vulgatus
Bifidobacteria and lactobacilli (or at least some members of these groups) are considered by some authors to be potentially protective against IBD.5,38–40
This is essentially based on studies with probiotic strains which appear to protect against inflammation in animal models of IBD5,38,39
and in recent clinical trials.9,40,41
One previous study suggested that bifidobacteria counts were decreased during CD,42
and we noted a trend towards such a decrease in our patients, the decrease being significant only in patient with inactive disease. The putative protective role of bifidobacteria against CD remains to be confirmed, but Campieri et al
reported that the probiotic VSL#3 which contains four strains of bifidobacteria decreased the risk of postoperative recurrence of CD.9
Interestingly, we found that a high proportion of the endogenous microbiota (approximately 30%) in patients with CD did not belong to the dominant microbiota of healthy controls. Identification of the bacterial species or groups present in this “phylogenetic gap” could be important for understanding of the pathogenesis of CD. A description of the entire bacterial population of patients with CD, based on cloning and sequencing of 16S rDNA,23
is currently underway.
The TTGE profile of the faecal microflora was very stable over time under healthy conditions but unstable in patients. We observed only a slight decrease in the number of bands during the active phase of the disease, showing that the microflora retains a high degree of diversity in both situations. We observed no specific single band pointing to the presence of bacteria which could be specifically involved in disease activity.
A better understanding of the alterations in the microflora should help to identify therapeutic targets for antibiotics, probiotics, and prebiotics. Our results suggest that antibiotics targeting enterobacteria or bacteria responsible for the phylogenetic gap should be studied in patients with CD. Among probiotics, those containing bifidobacteria should receive special attention in trials to prevent relapse of colonic CD.