腸躁症（IBS）與發炎性腸道疾病（IBD）的生物膜：深入解析、診斷與治療前景

Gastroenterology. 2021 Oct;161(4):1245–1256.e20. doi: 10.1053/j.gastro.2021.06.024

Mucosal Biofilms Are an Endoscopic Feature of Irritable Bowel Syndrome and Ulcerative Colitis

Maximilian Baumgartner 1, Michaela Lang 1,2, Hunter Holley 1,2, Daniel Crepaz 2, Bela Hausmann 3,4, Petra Pjevac 2,3, Doris Moser 5, Felix Haller 1, Fabian Hof 1, Andrea Beer 6, Elisabeth Orgler 1, Adrian Frick 1, Vineeta Khare 1, Rayko Evstatiev 1, Susanne Strohmaier 7, Christian Primas 1, Werner Dolak 1, Thomas Köcher 8, Kristaps Klavins 9, Timo Rath 10, Markus F Neurath 10, David Berry 2,3, Athanasios Makristathis 3,4, Markus Muttenthaler 11,12, Christoph Gasche 1,13,∗

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PMCID: PMC8527885  PMID: 34146566

Abstract

Background & Aims

Irritable bowel syndrome (IBS) and inflammatory bowel diseases result in a substantial reduction in quality of life and a considerable socioeconomic impact. In IBS, diagnosis and treatment options are limited, but evidence for involvement of the gut microbiome in disease pathophysiology is emerging. Here we analyzed the prevalence of endoscopically visible mucosal biofilms in gastrointestinal disease and associated changes in microbiome composition and metabolism.

Methods

The presence of mucosal biofilms was assessed in 1426 patients at 2 European university-based endoscopy centers. One-hundred and seventeen patients were selected for in-depth molecular and microscopic analysis using 16S ribosomal RNA gene amplicon-sequencing of colonic biopsies and fecal samples, confocal microscopy with deep learning–based image analysis, scanning electron microscopy, metabolomics, and in vitro biofilm formation assays.

Results

Biofilms were present in 57% of patients with IBS and 34% of patients with ulcerative colitis compared with 6% of controls (P < .001). These yellow-green adherent layers of the ileum and right-sided colon were microscopically confirmed to be dense bacterial biofilms. 16S-sequencing links the presence of biofilms to a dysbiotic gut microbiome, including overgrowth of Escherichia coli and Ruminococcus gnavus. R. gnavus isolates cultivated from patient biofilms also formed biofilms in vitro. Metabolomic analysis found an accumulation of bile acids within biofilms that correlated with fecal bile acid excretion, linking this phenotype with a mechanism of diarrhea.

Conclusions

The presence of mucosal biofilms is an endoscopic feature in a subgroup of IBS and ulcerative colitis with disrupted bile acid metabolism and bacterial dysbiosis. They provide novel insight into the pathophysiology of IBS and ulcerative colitis, illustrating that biofilm can be seen as a tipping point in the development of dysbiosis and disease.

Keywords: Endoscopy, Microbiota, Functional Gastrointestinal Disorders, Bacterial–Epithelial Interaction

Abbreviations used in this paper: ASV, amplicon sequencing variant; BA, bile acid; BF–, biofilm negative; BF+, biofilm-positive; DAPI, 4′,6-diamidino-2-phenylindole; GI, gastrointestinal; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; OTU, operational taxonomic unit; PAS, periodic acid-Schiff; PEG, polyethylene glycol; rRNA, ribosomal RNA; SEM, scanning electron microscopy; UC, ulcerative colitis; UCDA, ursodeoxycholic acid

Graphical abstract

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Bacterial biofilms were observed by colonoscopy as yellow-green membranous layers on the mucosa of the small and large intestinal junction and are specifically prevalent in irritable bowel syndrome and inflammatory bowel disease.

What You Need to Know.

Background and Context

IBS is the most common digestive disorder, affecting up to 15% of the Western population. Involvement of the microbiome in disease pathogenesis has been suggested, as fecal microbiota transplantation leads to symptom improvement.

New Findings

Previously unrecognized endoscopically visible biofilms are attached to the mucosa of the ileum and right colon in almost two-thirds of patients with IBS and one-third of patients with UC. They are associated with dysbiosis (ie, overgrowth of Escherichia coli and Ruminococcus gnavus) and increased fecal bile acid excretion.

Limitations

This is the first report on such biofilms observed by colonoscopy from 2 tertiary university-based teaching hospitals. To this point, mechanistic studies on the pathogenicity of such biofilms in gastrointestinal homeostasis are limited. Interventional studies on disruption of biofilms are needed to establish a causative involvement in IBS.

Impact

As these biofilms are associated with alterations of microbiota and bile acid metabolism, they may be involved in disease pathogenesis. For the clinician, visualization of biofilms by colonoscopy may provide a new diagnostic characteristic of IBS and disruption of such biofilms may offer a novel treatment path.

Irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBDs) affect 10%–15% and 0.5%–1% of the Western population, respectively, with the prevalence of both increasing worldwide.1,2 Patients with IBS have recurrent abdominal pain and changes in stool habits, but lack obvious signs of gastrointestinal (GI) inflammation. Ulcerative colitis (UC) and Crohn’s disease are the most prevalent forms of IBD and are characterized by a prolonged, debilitating inflammation of the GI tract, leading to abdominal pain, diarrhea, intestinal blood loss, and anemia. Such symptoms are associated with a substantial reduction in quality of life, as well as a considerable socioeconomic impact with high hospitalization costs.3 Although IBDs are diagnosed by endoscopy, no such immediate diagnostic test exists for IBS. Many patients with IBS are disappointed with current symptomatic medical care and lack of a causative treatment approach.4 Western lifestyle, including frequent antibiotic therapy and microbiota-altering food additives, have been implicated in disease development.5,6 Recently, alterations in bacterial bile acid (BA) metabolism have come into focus in IBS pathophysiology.7,8 Transplantation of fecal matter from healthy donors leads to a transient improvement of IBS symptoms.9,10 Changes in the relative abundance of bacterial taxa have been observed via high-throughput sequencing,11, 12, 13 but research on bacterial biomass or the spatial distribution of bacterial communities remains limited.

Biofilm formation is a distinct microbial mode of growth in which adherent prokaryotic communities embed themselves in a complex extracellular matrix to obtain competitive advantages. Biofilm-forming bacteria predominate numerically and metabolically in virtually all ecosystems, and are also involved in chronic bacterial infections of the human body.14,15 While in a healthy gut bacterial growth is usually scattered as small microcolonies,16,17 polymicrobial biofilms have been observed microscopically in IBD, GI infections, right-colonic cancer, and familial adenomatous polyposis.18, 19, 20, 21, 22 However, a macroscopically visible aspect of biofilm formation in the intestine has never been considered. Stressors on the microbiota, such as overactivation of the immune system in IBDs,23 chronic use of microbiome-altering pharmaceuticals24 (including immunosuppressive medication, proton pump inhibitors, or recurrent use of antibiotics), and food additives (eg, with antimicrobial and/or detergent activity), as well as GI infections and excessive hygiene,25 lead to selection pressures that might trigger microbial defense mechanisms, such as orchestrated biofilm formation.26

In this work, we systematically studied 2 endoscopy cohorts with a total of 1426 patients, demonstrating that regularly observed yellow-green adherent layers of the ileum and right-sided colon are indeed biofilms that are readily visible during high-definition white light endoscopy. Such biofilms are highly prevalent in IBS, to a lesser extent in IBDs, and in a post-organ transplantation cohort. We further applied a range of multidisciplinary techniques including 16S ribosomal RNA (rRNA) gene amplicon sequencing, scanning electron microscopy (SEM), confocal microscopy with deep learning–based image analysis, in vitro biofilm formation assays, and metabolomics to characterize these biofilms. We thereby provide advanced understanding of their origin and novel opportunities for future diagnosis and treatment options.