Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease - PubMed (original) (raw)

Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease

Alexander Swidsinski et al. J Clin Microbiol. 2005 Jul.

Abstract

The composition and spatial organization of the mucosal flora in biopsy specimens from patients with inflammatory bowel disease (IBD; either Crohn's disease or ulcerative colitis), self-limiting colitis, irritable-bowel syndrome (IBS), and healthy controls were investigated by using a broad range of fluorescent bacterial group-specific rRNA-targeted oligonucleotide probes. Each group included 20 subjects. Ten patients who had IBD and who were being treated with antibiotics were also studied. Use of nonaqueous Carnoy fixative to preserve the mucus layer was crucial for detection of bacteria adherent to the mucosal surface (mucosal bacteria). No biofilm was detectable in formalin-fixed biopsy specimens. Mucosal bacteria were found at concentrations greater than 10(9)/ml in 90 to 95% of IBD patients, 95% of patients with self-limiting colitis, 65% of IBS patients, and 35% of healthy controls. The mean density of the mucosal biofilm was 2 powers higher in IBD patients than in patients with IBS or controls, and bacteria were mostly adherent. Bacteroides fragilis was responsible for >60% of the biofilm mass in patients with IBD but for only 30% of the biofilm mass in patients with self-limiting colitis and <15% of the biofilm mass in patients with IBS. In contrast, bacteria which positively hybridized with the probe specific for Eubacterium rectale-Clostridium coccoides accounted for >40% of the biofilm in IBS patients but for <15% of the biofilm in IBD patients. In patients treated with (5-ASA) or antibiotics, the biofilm could be detected with 4,6-diamidino-2-phenylindole but did not hybridize with fluorescence in situ hybridization probes. A Bacteroides fragilis biofilm is the main feature of IBD. This was not previously recognized due to a lack of appropriate tissue fixation. Both 5-ASA and antibiotics suppress but do not eliminate the adherent biofilm.

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Figures

FIG. 1.

FIG. 1.

Three adjacent microscopic fields of the ascending colon of an untreated CD patient show a biofilm containing adherent Bacteroides fragilis (visualized with the Bfra-Cy3 probe). The biofilm completely covers the mucosal surface and enters the crypts. The epithelial tissue structures are not stained; however, they are well visualized due to autofluorescence.

FIG. 2.

FIG. 2.

Triple-color FISH identifies organisms present in biofilms covering the mucosae in patients with CD (left), self-limiting colitis (middle), and IBS (right). Bacteroides fragilis (Bfra-Cy3 probe) appears yellowish on a green background; the Eubacterium rectale group (Erec-Cy5 probe) appears dark red. All other bacteria that hybridize exclusively with the universal probe (probe Eub-FITC) appear green. There is a striking increase in Bacteroides fragilis concentrations from patients with IBS to patients with IBD, along with changes in bacterial localization.

FIG. 3.

FIG. 3.

Three photos of the same microscopic fields made at different focus levels. Intracellular bacteria (Eub-Cy3 probe) are located in a single epithelial cell at the bottom of the crypt.

FIG. 4.

FIG. 4.

Sigmoid colon biopsy specimen from an UC patient treated with 3 g mesalamine orally plus 4-g mesalamine enemas. Only a small number of bacteria located within crypts definitively hybridized with the Eub338 probe universal for bacteria (Cy3 orange fluorescence is on the left). The same microscopic view with DAPI fluorescence revealed a thin but tightly adherent biofilm that was not amenable to FISH probes, that covered the biopsy surface, and that extended deep into the crypt.

FIG. 5.

FIG. 5.

Sigmoid colon biopsy specimen from a CD patient who had received antibiotics (metronidazole and ciprofloxacin) the day prior to the colonoscopy. The bacterial biofilm is still seen with the DAPI stain (right) but is not accessible to FISH probes (Eub338 probe, left).

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References

    1. Amann, R., L. Krumholz, and D. A. Stahl. 1990. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172:762-770. - PMC - PubMed
    1. Amann, R., J. Stromley, R. Devereux, R. Key, and D. A. Stahl. 1992. Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl. Environ. Microbiol. 58:614-623. - PMC - PubMed
    1. Amann, R. I., W. Ludwig, and K.-H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143-169. - PMC - PubMed
    1. Bohnert, J., B. Hübner, and K. Botzenhart. 2002. Rapid identification of Enterobacteriaceae using a novel 23S rRNA-targeted oligonucleotide probe. Int. J. Hyg. Environ. Health 203:77-82. - PubMed
    1. Chapman, R. W., W. S. Selby, and D. P. Jewell. 1986. Controlled trial of intravenous metronidazole as an adjunct to corticosteroids in severe ulcerative colitis. Gut 27:1210-1212. - PMC - PubMed

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