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- Materials and Methods
Background: The mucosa-associated microbiota, being very close to the inflammatory process associated with inflammatory bowel disease (IBD), may have a pathogenic role. We used a culture-independent method to analyze the mucosa-associated microbiota in IBD patients at various points of the distal digestive tract.
Methods: Thirty-five patients (20 with Crohn's disease, 11 with ulcerative colitis, and 4 controls) underwent colonoscopy. Biopsies (n = 126) were taken from 4 sites: the ileum, right colon, left colon, and rectum. Fecal samples were also obtained from 7 individuals. Temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA was used to evaluate dominant species diversity. TTGE profiles were compared using software that measures the degree of similarity.
Results: In a given individual, the overall similarity percentage between the 4 segments of the distal digestive tract was 94.7 ± 4.0%, regardless of clinical status. The average similarity of all profiles for a given segment was 59.3 ± 18.3% in the overall population. Dendrogram analysis showed that TTGE profiles did not cluster with clinical status. Differences were observed between the dominant fecal microbiota and the mucosa-associated microbiota of all 4 sites, with similarity percentages less than 92%.
Conclusions: These results confirm that the dominant species differ between the mucosa-associated and fecal microbiota. They also show that, in a given individual, the microbiota is relatively stable along the distal digestive tract, showing a slight evolution in dominant species diversity from the ileum to the rectum, in both healthy subjects and patients with IBD.
The endogenous microbiota plays an important role in modulating the mucosal immune response and in the pathogenesis of inflammatory bowel disease (IBD), both in animal models and in humans.1–4 The microbiota is a growing focus of interest in patients with Crohn's disease (CD) and ulcerative colitis (UC). The advent of culture-independent bacteriological methods has facilitated microbiota analysis.5–8 Several clinical and experimental studies showed that the luminal biota plays an important role in the pathogenesis of CD. For example, diversion of the fecal stream prevented postoperative recurrence of ileal CD.9 Likewise, Harper et al10 reported that reintroduction of small bowel effluent into the intestinal lumen of patients with CD treated by split ileostomy triggered inflammation, whereas reintroduction of a sterile ultrafiltrate of the effluent did not. HLA-B27/human α2 microglobulin transgenic rats and interleukin 2 (IL-2) and IL-10 knock-out mice developed spontaneous intestinal inflammation, whereas inflammation was absent or attenuated when animals were kept in a germ-free state.11–17 In a recent study, Rakoof-Nahoum et al18 reported that commensal bacteria were recognized by Toll-like receptors (TLR) under normal conditions. Interactions of commensal bacterial products with those microbial pattern recognition receptors played a critical role in resistance to epithelial injury and in intestinal homeostasis. Thus, a dysregulated interaction between bacteria and TLR may promote chronic inflammation. We and others have previously reported that the fecal microbiota of patients with IBD differed from that of healthy subjects,19–22 containing more Enterobacteriaceae and more bacteria unusually found among the dominant biota,23 and that the biodiversity of this ecological niche remained high during the disease.19 The microbiota close to the mucosa, which differs from the luminal microbiota,24 has so far received less attention, yet, it is very close to the inflammatory process.25 Culture-based studies have led to the isolation, from early and chronic ileal CD lesions, of a new pathovar of Escherichia coli, designated adherent-invasive E. coli (AIEC), that may colonize the intestinal mucosa by adhering to intestinal epithelial cells.26–28 This adherent E. coli strain is specifically associated with ileal mucosa in CD and could be involved in the initiation of the inflammatory process.29 Rayment et al30 suggested an imbalance between Bacteroides and Clostridium spp. on the one hand and Bifidobacterium and Lactobacillus spp. on the other hand in the rectal mucosa-associated microbiota of patients with active IBD, whereas Swidsinski et al,31 using both culture and culture-independent methods, reported no major difference in the composition of the mucosal microbiota between patients with IBD and controls. Bifidobacteria and peptostreptococci have also been implicated in UC.32
The aim of this study was to analyze the mucosa-associated microbiota in patients with IBD at various sites of the colon and ileum. We chose to use temporal temperature gradient gel electrophoresis (TTGE), a culture-independent method, because it allows the dominant species diversity to be compared among samples.
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- Materials and Methods
This study confirms that the dominant mucosa-associated microbiota differs from the dominant fecal microbiota. Furthermore, it shows that, in a given individual, the microbiota is relatively stable along the distal digestive tract, showing a slight evolution in dominant species diversity from the ileum to the rectum, in both healthy subjects and patients with active or inactive CD.
The method used here-denaturing gel electrophoresis-is capable of separating bacterial sequences with the same size but different thermal stability.19,35 Because 16S rDNA from different bacterial species have different nucleotide sequences in variable regions, their thermal stability is also different. In theory, sequences differing by a single base can be separated by this method. When applied to complex microbial communities, this method gives profiles corresponding to all the dominant bacterial species present in the sample. This study did not aim to determine the composition of the mucosa-associated microbiota in terms of bacterial genera or species but assessed for each individual the biodiversity along the digestive tract. The method used was very efficient for comparing the biodiversity of complex bacterial communities. TTGE profiles were obtained with 120 of the 126 biopsy specimens tested in this study. Five of the 6 biopsy specimens that yielded less than 3 bands after PCR were from patients with acute phase IBD. This PCR amplification failure could be caused by a reduced bacterial biomass or the presence of PCR inhibitors. Previous studies have shown an increased mucosal bacterial load in IBD patients relative to healthy controls,31,37 supporting the second explanation. However, the differences in mucosal biomass between published results may partly be caused by different sample preparations. Indeed, a significant part of the intestinal microbiota may be associated with mucus,37 and some authors thoroughly wash biopsy specimens before analysis.31 In our study, no biopsy wash was performed to take into account the bacteria of the mucus layer. Furthermore, a wash step for biopsies could have a detrimental effect on anaerobic bacteria, thus underestimating the microbiota diversity. The patients in our control group were older than our IBD patients (60 versus 39 yr). Indeed, full colonoscopy is rarely indicated in young subjects without IBD. Several studies38–41 have shown that the intestinal microbiota differs in elderly subjects more than 65 years of age, and precise knowledge is presently lacking concerning potential changes in adults more than 40 years of age. The effects of dietary factors such as consumption of prebiotics or fermented milks containing probiotics on the mucosal microbiota are currently assessed by many research teams in the hope to modify the course of IBD.1,42 We did not study dietary habits, which probably differed between our patient groups. Indeed, this study focused on the stability of the microbiota between different parts of the distal intestinal tract in an individual but did not intend to identify modulating factors.
In a given individual, most of the bands obtained with the samples from the ileum, right colon, left colon, and rectum were common with similar intensities. This indicates that essentially the same bacterial species predominate from the ileal to the rectal mucosa. Similar results have been obtained in the colon by Zoetendal et al.24 The ecological conditions in the intestinal lumen differ greatly between the ileum, right colon, and distal colon. We and others have previously reported that the luminal biota also differs greatly between these parts of the distal digestive tract.43,44 Therefore our results strongly suggest that the composition of the mucosa-associated microbiota is more strongly influenced by host factors than by environmental conditions. The mucosa-associated microbiota might have a more important pathogenic role than the luminal microbiota in some intestinal diseases, because it is closer to epithelial and immune cells. Increased adherence of commensal bacteria to inflamed mucosa may enhance the exposure of the mucosal immune system to intestinal bacteria or bacterial components, resulting in sustained inflammation.37 In this study, we also found very few regional differences in the mucosa-associated microbiota in patients with IBD, whatever their clinical status. These differences were mostly observed in the inactive and active CD groups. We observed a trend in the dominant bacterial species from the rectum to the ileum. The rectal microbiota was more similar to the left colon one than to the right colon or ileal one. Moreover, the microbiota associated to the right colon mucosa was closer to the left colon one than to the rectal one in both CD groups. The same tendency was observed for the control group, but the number of comparisons was too small to be statistically analyzed.
The mucosa-associated microbiota and fecal microbiota appeared different. This confirms the result obtained by Zoetendal et al24 in healthy individuals, and the significance was also confirmed despite the low number of patients (n = 7). Moreover, the mucosa-associated microbiota was significantly more similar to the fecal microbiota in patients with active CD than in those with active UC (P < 0.05). This may suggest that bacterial agents present in the luminal biota have a more important pathogenic role in CD than in UC.
Our results also showed that the mucosa-associated microbiota was specific to each individual. This is consistent with results reported for fecal samples.19,35 The average similarity percentage among the 30 patients studied was 59.3 ± 18.3%. Previously, Mangin et al23 studied the microbial species composing the fecal microflora of patients with CD using a molecular inventory method. They showed numerous uncommon clones in the feces of those patients. This would lead to an increase in interindividual variability, which is consistent with our observations in the patients with IBD. Indeed, we found the microbiota associated to the left colon to be more similar between healthy individuals than between CD patients. In addition, the rectal mucosa-associated microbiota was also more similar in healthy controls than between CD or UC patients. In contrast, dendrogram analysis did not separate the profiles according to clinical status, suggesting that the dominant mucosal microbiota is not specifically altered by IBD, regardless of disease activity. If the mucosa-associated microbiota is indeed involved in pathogenesis of IBD, we identified no dominant bacterial species specific to either CD or UC. Moreover, the high complexity of the dominant mucosa-associated microbiota, as reflected by TTGE profiles, does not suggest that a single group of bacteria is involved. Culture-based studies have also shown marked interindividual variability of the microbiota in terms of both total counts and genera present in rectal biopsy specimens from patients with UC.45,46
In conclusion, we observed, both in individuals with IBD and in healthy controls, a remarkably stable dominant mucosa-associated microbiota from the ileum to the rectum. A slight trend toward higher similarities was noticed between adjacent segments. No particular pattern of dominant mucosa-associated microbiota was linked to the disease status or location. This suggests that microbial factors possibly involved in the pathogenesis of IBD are likely to be complex, in keeping with the concept of dysbiosis.47 Our work emphasized that the mucosa-associated microbiota differed from the fecal microbiota, and this should be taken into account in future studies on the role of the endogenous microbiota in IBD. In addition, because the mucosa-associated microbiota seems to be specific of an individual, it would be highly relevant to compare, for each patient with CD, ulcerated mucosa to nonulcerated mucosa, the latter being considered as an internal control.