Colon ORIGINAL RESEARCH Gut: first published as 10.1136/gutjnl-2020-321466 on 11 November 2020. Downloaded from Association between Brachyspira and irritable bowel syndrome with diarrhoea Karolina S Jabbar,1,2 Brendan Dolan,1 Lisbeth Eklund,1,2 Catharina Wising,1 Anna Ermund,1 Åsa Johansson,1 Hans Törnblom,2,3 Magnus Simren,2,3 Gunnar C Hansson 1 ► Additional material is ABSTRACT published online only. To view, Objective The incidence of IBS increases following Significance of this study please visit the journal online enteric infections, suggesting a causative role for (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2020- 321466). microbial imbalance. However, analyses of faecal microbiota have not demonstrated consistent alterations. ► IBS incidence increases after enteric infections, 1Department of Medical Here, we used metaproteomics to investigate potential suggesting a possible causative role for Biochemistry, Institute of microbial perturbation. Biomedicine, University of associations between mucus-resident microbiota and IBS Gothenburg, Gothenburg, symptoms. ► Studies of the faecal microbiota have failed to Sweden Design Mucus samples were prospectively collected demonstrate consistent alterations associated 2Department of with IBS symptoms. Gastroeneterology and from sigmoid colon biopsies from patients with Hepatology, Sahlgrenska IBS and healthy volunteers, and their microbial What are the new findings? University Hospital, Gothenburg, protein composition analysed by mass spectrometry. ► This study represents the first comparison of Sweden Observations were verified by immunofluorescence, 3 the microbial community of the colonic inner Department of Internal electron microscopy and real-time PCR, further confirmed Medicine and Clinical Nutrition, mucus layer of patients with IBS and healthy Institute of Medicine, University in a second cohort, and correlated with comprehensive volunteers. of Gothenburg, Gothenburg, profiling of clinical characteristics and mucosal immune ► Colonisation of the colonic epithelial surface or Sweden responses. mucus layers by pathogenic Brachyspira species Results Metaproteomic analysis of colon mucus was detected in 40% of patients with IBS with Correspondence to samples identified peptides from potentially pathogenic diarrhoea but not in any healthy individual. Dr Gunnar C Hansson, Dept. Brachyspira species in a subset of patients with IBS. Medical Biochemistry, University ► Brachyspira- associated IBS was linked to of Gothenburg, Gothenburg 405 Using multiple diagnostic methods, mucosal Brachyspira distinctive clinical, histological and molecular 30, Sweden; colonisation was detected in a total of 19/62 (31%) characteristics, suggesting that it should be http://gut.bmj.com/ gunnar. hansson@ medkem. gu. se patients with IBS from two prospective cohorts, versus considered a separate diagnostic entity. 0/31 healthy volunteers (p<0.001). The prevalence of MS and GCH are joint senior ► Treatment paradoxically resulted in relocation authors. Brachyspira colonisation in IBS with diarrhoea (IBS-D) of the Brachyspira into goblet cell secretory was 40% in both cohorts (p=0.02 and p=0.006 vs granules. Received 17 April 2020 controls). Brachyspira attachment to the colonocyte Revised 12 October 2020 apical membrane was observed in 20% of patients with How might it impact on clinical practice in the Accepted 13 October 2020 foreseeable future? IBS and associated with accelerated oro-anal transit, on October 1, 2021 by guest. Protected copyright. mild mucosal inflammation, mast cell activation and ► The presence of Brachyspira may be used alterations of molecular pathways linked to bacterial to identify a distinct subset of patients with uptake and ion–fluid homeostasis. Metronidazole IBS, who could potentially be responsive to treatment paradoxically promoted Brachyspira relocation eradication therapy. into goblet cell secretory granules—possibly representing ► The relocation of the Brachyspira into goblet a novel bacterial strategy to evade antibiotics. cell mucus granules likely represents a novel Conclusion Mucosal Brachyspira colonisation was bacterial strategy to evade antibiotics, which significantly more common in IBS and associated could inform our understanding of other with distinctive clinical, histological and molecular persistent or recurrent mucosal infections. characteristics. Our observations suggest a role for © Author(s) (or their Brachyspira in the pathogenesis of IBS, particularly IBS-D . employer(s)) 2020. Re- use Gut microbiota composition studies over- permitted under CC BY- NC. No whelmingly rely on faecal material. However, commercial re- use. See rights and permissions. Published these samples largely reflect the luminal microbial by BMJ. INTRODUCTION community, which is spatially separated from the The incidence of IBS steeply increases following a colonic epithelium and underlying immune cells To cite: Jabbar KS, Dolan B, gastroenteritis episode, suggesting a possible caus- through a two- tiered mucus barrier.4 By contrast, Eklund L, et al. Gut Epub 1 ahead of print: [please ative role for microbial perturbation. Still, previous certain species have found a niche in the outer include Day Month Year]. investigations have not demonstrated consistent mucus layer, feeding on the abundant mucin O- gly- doi:10.1136/ associations between intestinal microbiota and IBS cans.5 Thus, faecal and mucus-associated bacteria gutjnl-2020-321466 symptoms.2 3 represent distinctive populations, with the latter Jabbar KS, et al. Gut 2020;0:1–13. doi:10.1136/gutjnl-2020-321466 1 Colon Gut: first published as 10.1136/gutjnl-2020-321466 on 11 November 2020. Downloaded from Figure 1 Graphical summary of the study design. Schematic representation of the different phases of the study. In-depth profiling of clinical, histological and molecular characteristics (phase 4) was performed in participants where Brachyspira colonisation could be confirmed/rejected with high confidence, typically based on consistent results from two different methods. Quantification of mucosal immune cells by histology was performed http://gut.bmj.com/ in a representative subset of participants with good-quality biopsy sections, whereas analysis of the human mucus proteome was largely restricted to participants from the explorative cohort. The figure was created with BioRender.com. more likely to influence the epithelium. In particular, bacterial analysis (figure 1). Carnoy’s fixative enables optimal preser- 6 presence in the inner mucus layer might result in epithelial stress vation of both the mucus layers and the tissue nucleic acids. and immune activation. Here, we performed a metaproteomic Comprehensive clinical characterisation, including assessment of on October 1, 2021 by guest. Protected copyright. analysis of inner mucus layer samples from patients with IBS and oro- anal transit time (OATT) and rectal sensitivity, was under- healthy individuals, to investigate potential associations between taken in patients with IBS. mucus-resident microbiota and IBS symptoms. In a randomly selected subset of participants (the first/explor- ative cohort, IBS n=22, healthy n=14), mucus was collected MATERIALS AND METHODS from ex vivo sigmoid colon biopsies and analysed by mass spec- Study design trometry (MS). The objectives of this explorative phase were Patients aged 18–65 years fulfilling the Rome III criteria for twofold: to characterise the metaproteomic composition of IBS were prospectively included at a secondary/tertiary care the inner mucus layer of patients with IBS and healthy individ- unit (Sahlgrenska Hospital, Gothenburg, Sweden) June 2012 uals, and to generate hypotheses to pursue in the entire study through April 2018. Healthy volunteers were recruited through population. Patients in whose mucus/tissue samples bacteria advertisements and screened by questionnaires and interviews were detected by MS (n=9), or, in certain cases, other methods to rule out gastrointestinal complaints and comorbidities. Exclu- (n=3), underwent repeat sigmoidoscopy, with biopsy sampling sion criteria are listed in the online supplemental file. Patients for electron microscopy, mucus collection and/or routine clinical with IBS were subtyped based on the Rome III criteria and the histopathology. Bristol Stool Form scale, as follows: IBS with diarrhoea (IBS- The metaproteomic analysis identified a putative link between D), IBS with constipation (IBS-C), IBS with mixed bowel habits the Brachyspira genus and IBS. This association was validated (IBS- M) and unsubtyped IBS without diarrhoea or constipation in the explorative cohort, as well in the remainder of the study (IBS- U). All participants provided written informed consent. population—the second cohort, comprising 40 patients with IBS All study participants (IBS n=62, healthy n=31) underwent and 17 healthy individuals—using real- time PCR and immuno- sigmoidoscopy with sampling of biopsies in methanol-Carnoy fluorescence. Sampling and analysis were, whenever possible, for future histology/immunohistochemistry and real-time PCR performed with blinding to participant identity. Missing data 2 Jabbar KS, et al. Gut 2020;0:1–13. doi:10.1136/gutjnl-2020-321466 Colon due to technical failures or suboptimal biopsy specimens are Transmission electron microscopy tabulated in online supplemental table S1. Transmission electron microscopy analysis of biopsies from 10 Gut: first published as 10.1136/gutjnl-2020-321466 on 11 November 2020. Downloaded from participants