Gastroenterology

Gastroenterology

Volume 153, Issue 4, October 2017, Pages 1026-1039
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Intestinal Fungal Dysbiosis Is Associated With Visceral Hypersensitivity in Patients With Irritable Bowel Syndrome and Rats

https://doi.org/10.1053/j.gastro.2017.06.004Get rights and content

Background & Aims

Visceral hypersensitivity is one feature of irritable bowel syndrome (IBS). Bacterial dysbiosis might be involved in the activation of nociceptive sensory pathways, but there have been few studies of the role of the mycobiome (the fungal microbiome) in the development of IBS. We analyzed intestinal mycobiomes of patients with IBS and a rat model of visceral hypersensitivity.

Methods

We used internal transcribed spacer 1-based metabarcoding to compare fecal mycobiomes of 18 healthy volunteers with those of 39 patients with IBS (with visceral hypersensitivity or normal levels of sensitivity). We also compared the mycobiomes of Long-Evans rats separated from their mothers (hypersensitive) with non-handled (normally sensitive) rats. We investigated whether fungi can cause visceral hypersensitivity using rats exposed to fungicide (fluconazole and nystatin). The functional relevance of the gut mycobiome was confirmed in fecal transplantation experiments: adult maternally separated rats were subjected to water avoidance stress (to induce visceral hypersensitivity), then given fungicide and donor cecum content via oral gavage. Other rats subjected to water avoidance stress were given soluble β-glucans, which antagonize C-type lectin domain family 7 member A (CLEC7A or DECTIN1) signaling via spleen-associated tyrosine kinase (SYK), a SYK inhibitor to reduce visceral hypersensitivity, or vehicle (control). The sensitivity of mast cells to fungi was tested with mesenteric windows (ex vivo) and the human mast cell line HMC-1.

Results

α diversity (Shannon index) and mycobiome signature (stability selection) of both groups of IBS patients differed from healthy volunteers, and the mycobiome signature of hypersensitive patients differed from that of normally sensitive patients. We observed mycobiome dysbiosis in rats that had been separated from their mothers compared with non-handled rats. Administration of fungicide to hypersensitive rats reduced their visceral hypersensitivity to normal levels of sensitivity. Administration of cecal mycobiomes from rats that had been separated from their mothers (but not non-handled mycobiome) restored hypersensitivity to distension. Administration of soluble β-glucans or a SYK inhibitor reduced visceral hypersensitivity, compared with controls. Particulate β-glucan (a DECTIN-1 agonist) induced mast cell degranulation in mesenteric windows and HMC-1 cells responded to fungal antigens by release of histamine.

Conclusions

In an analysis of patients with IBS and controls, we associated fungal dysbiosis with IBS. In studies of rats, we found fungi to promote visceral hypersensitivity, which could be reduced by administration of fungicides, soluble β-glucans, or a SYK inhibitor. The intestinal fungi might therefore be manipulated for treatment of IBS-related visceral hypersensitivity.

Section snippets

Patient Characteristics and Ethics Statement

Patient characteristics are provided in Table 1. The IBS and healthy volunteer population included are subgroups of the Maastricht-IBS cohort, comprising 540 IBS patients and 205 healthy controls. The patients represent a mixed population from primary to tertiary care with an established clinical diagnosis of IBS according to the Rome III criteria. Age, gender, medication use, mean anxiety, depression, and symptom scores do not differ when compared with the total IBS cohort (data not shown).

Mycobiome Differences Exist Between Healthy Volunteers, Hypersensitive IBS, and Normally Sensitive IBS Patients

We compared the fecal mycobiome of healthy volunteers, hypersensitive IBS patients, and normally sensitive IBS patients. Figure 1A depicts the 30 most predominant species and shows that the human mycobiome is dominated by 2 yeasts; Saccharomyces cerevisiae and Candida albicans. In healthy volunteers, their combined presence was 57% of total reads. In IBS these species were even more predominant and added up to 76% and 83% in hypersensitive and normally sensitive IBS patients, respectively.

Discussion

Until now, investigations on the possible role of gut microbial communities in IBS almost exclusively focused on bacteria. Based on recent evidence that fungi can aggravate disease severity in colitis,19, 21, 22, 23, 24 we assessed the role of the gut mycobiome in IBS. We observed altered mycobiome composition in patients, which led us to assess the role of fungi in the maternal separation model for stress-induced visceral hypersensitivity. Fungicide treatment of hypersensitive rats reversed

Acknowledgements

Raw sequence data will be deposited in the European Nucleotide Archive.

Guus Roeselers, Evgeni Levin, and Daisy M. Jonkers contributed equally to is work.

The current affiliation for Guus Roeselers and Heleen H. de Weerd is: Danone-Nutricia Research, Utrecht, The Netherlands.

The current affiliations for Evgeni Levin is:Department of Experimental Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands; and Horaizon BV, Rotterdam, The Netherlands.

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    Conflicts of interest The authors disclose the following: W.deJ. has received consultancy fees from GlaxoSmithKline, receives research grant support from GlaxoSmithKline, Dr Willmar Schwabe GMbH, and Mead Johnsson Nutrition Pediatric Institute. R.vdW. receives research grant support from Dr Willmar Schwabe GMbH. All other authors disclose no competing financial interests.

    Funding S.B. was supported by The Netherlands Digestive Diseases Foundation, project no. 1CDP005. W.deJ. is supported by an NWO VIDI grant and Marie Curie ETN grant no. 641665.

    Author names in bold designate shared co-first authorship.

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