Bacterial strain colonization in fecal microbiota transfer therapy in patients with irritable bowel syndrome (STRACOL)

Functional gastrointestinal disorders such as irritable bowel syndrome (IBS), are poorly defined disorders characterized by prolonged abdominal pain combined with diarrhea or constipation of variable intensity [1]. These disorders are common in the population with around 12% of the Norwegian adult population reporting symptoms of IBS [2]. The illness puts a large demand on healthcare resource use, cause sick leave and have detrimental effects on the quality of life for those affected [3, 4].  There is a need for effective and sustainable treatments options. A study from 2014 found IBS patients willing to give up 10-15 years of their life expectancy for an immediate cure [5]. The mechanisms for development of these syndromes are not well understood, and there are no objective disease markers [6].  Available treatment options and dietary advice are only for symptom relief, and few patients experience current treatment as satisfactory.

Decreased richness and diversity of the gut microbiota correlate with increased IBS symptom severity [7, 8]. A systematic review of 22 studies showed that the family Enterobacteriaceae (phylum Proteobacteria), family Lactobacillaceae, and genus Bacteroides were increased in patients with IBS compared with controls, whereas Clostridiales, genus Faecalibacterium (including Faecalibacterium prausnitzii), and genus Bifidobacterium were decreased in patients with IBS [9]. 

These studies were done using 16S amplicon sequencing which can only give a broad overview of bacterial families present in the gut, and rarely below the genus level. Newer studies are using shotgun (metagenomic) sequencing allowing greater microbial resolution with identification of more genera of bacteria, archaea, viruses, and eukaryota, and enables analysis of putative functional genes that would have gone undetected using 16S.

Even metagenomic sequencing often cannot accurately classify species or strains, affecting their applicability to infer microbial persistence and transfer [10]. Strains within a species may differ in their metabolism and function, so it is important to be able to link specific sequences to specific bacterial strains.

The human microbiota is increasingly studied as a diagnostic, therapeutic and disease prevention target. A particularly strong indication of the therapeutic potential of the colon microbiota is the use of fecal microbiota transplantation (FMT). FMT is an emerging treatment whereby donor feces are implanted in a patient’s intestinal tract. There is emerging evidence it may be effective for other diseases, including inflammatory bowel disease (IBD). A systematic review of 6 trials involving 324 patients with ulcerative colitis found that compared with placebo, FMT had significant short-term benefit in inducing combined clinical and endoscopic remission [11].

FMT for treating IBS has gained much attention recently. Indeed, five out of seven placebo-controlled trials have returned promising results regarding symptom reduction and gut microbiota recovery. Patient associations such as the Norwegian Mage-tarmforbundet has actively encouraged prioritization of research in this field. 
In Norway two randomised controlled studies of FMT in IBS have been conducted recently; one at one at Helse-Vest (HUS/Stord) and one at Helse-Nord (UNN/Harstad). At Helse Vest, Magdy ElSalhy et al reported the first placebo-controlled RCT in 165 IBS patients where FMT (30g FMT and 60g FMT in 40 mL saline) was installed via gastroscopy in the distal duodenum was compared with placebo. The investigation included 165 patients with IBS. Symptom remission, defined as IBS-SSS score improvement of at least 175 points, was achieved in 35% of patients receiving 30g of stool, 47% of patients receiving 60g of stool, and 6% of patients receiving placebo [12]. A follow-up study in 125 of these patients after 3 years showed that patients in the 30g and 60g groups had significantly fewer IBS symptoms, fatigue, and a greater quality of life both at 2 and 3 years after FMT [8]. 

The REFIT pilot trial was a phase II trial where 90 IBS patients were randomized to one donor FMT or autologous FMT instillation delivered by colonoscopy in a double blind, randomized, placebo controlled trial at Universitetssykehuset Nord-Norge (UNN). This study found that donor FMT significantly improved quality of life by the Irritable Bowel Syndrome Quality of Life questionnaire (IBS-QoL) (85% vs 61%) and fatigue by the Fatigue Impact Scale (FIS) (35% vs 11%) six months later.

The REFIT pilot trial encouraged the REFIT2 trial, an ongoing phase III multi-centre trial where Haukeland University Hospital (HUH) is involved. Enrolment is now finished, and an impressive biobank has been established.

Analysis of participants feces before and after treatment in the REFIT trial, and in the trial by ElSalhy et al, showed that symptom relief after FMT was mirrored by alterations in the microbiota [7, 8], including functional capabilities. Recipients responding to treatment seem to acquire a microbiota profile which is closer to the donor profile. Analysis in the REFIT study showed that 7 bacterial entities decreased while 25 increased after treatment [7]. A fecal composition analysis in the trial by ElSalhy et al found that microbiota dysbiosis index was decreased in the active treatment group with 10 probed bacterial genera/families/species showing significant correlations with IBS symptoms and fatigue after FMT. Interestingly, both these two Norwegian studies found a correlation with IBS symptom improvement and abundance of bacteria several bacterial genera, and families [7, 8].

The roles that these bacterial families play in IBS symptom manifestation are yet to be determined as the current methods often do not have the granularity to track specific species, or strains associated with therapeutic effect. As an example, abundance of bacteria in the Alistipes family has been shown to be associated with several diseases, such as depression, anxiety, chronic fatigue syndrome, autism, cirrhosis, and aging [13].  The specific Alistipes species or strains mediating this effect are not known.  The study by El-Salhy et al. found that levels of butyric acid, a beneficial short chain fatty acid, were significantly increased at one month and one year after donor FMT [14]. While both Faecalibacterium prausnitzii and Eubacterium biforme are important butyrate producers in the human colon [15] the transfer of specific donor strains of these bacterial species could not be ascertained. 

It is unlikely that therapeutic effect is mediated by single strain engraftment. Probably communities of several strains, positively interacting with each other, are needed for successful engraftment, and for beneficial function in a new host.

FMT has now been tested in many studies involving several thousand people for different conditions. Although results have differed by targeted disease and mode of delivery, it is a safe and well-tolerated therapy. Many studies have shown that IBS patients have lower diversity of their microbiota, and FMT has been seen to restore this diversity in many patients.

Still, our understanding of the mechanisms at play in FMT is very limited. Important unanswered questions are:

1. ​Which exact elements of the microbiota mediates the therapeutic effect?
2. Why does donor microbiota engraft in some patients but not in others?
   

In this proposal we focus on engraftment ability of donor bacterial strains and explore factors that influence engraftment success. The biobank of the REFIT2 trial is a solid opportunity to examine donor strain engraftment of specific bacterial strains in detail, and to increase our knowledge about factors for engraftment success. This understanding, and resulting microbe collection can open a path to development of a simplified microbial composition that can stably and more reliably improve symptoms and quality of life in patients with IBS.

The project group will be led by infectious diseases specialist and professor Kurt Hanevik, at the Medical department (HUH) and UiB.  He has performed studies on post-infectious IBS and chronic fatigue following the Giardia outbreak in Bergen, and coordinated the development of a human experimental model for enterotoxigenic E. coli. 

The project requires competencies in diverse but complementary scientific fields, including clinical medicine, microbiology, immunology and bioinformatics. Essential competencies among the partners include clinical studies (UiB, UNN, HUS), microbiology (UiB/HUH), bioinformatics (UiT/HUH) and immunology (HUS/UiB).  The proposed study will be performed in close collaboration with REFIT 2 researchers regarding biobank material and clinical data analysis including Rasmus Goll and Peter Holger Johnsen (UNN) and Jan Hatlebakk at gastroenterology department and «Nasjonal kompetansetjeneste for funksjonelle mage-tarmsykdommer» at Haukeland University Hospital (HUH).

Facilities for media production, anaerobic culture and MALDI-TOF mass spectrometer for screening colonies are available in collaboration with Dag Harald Skutlaberg at the Microbiology Department and the Geomicrobiology laboratory at UiB.  A technician at PhD level with experience in anaerobic culture will be hired. Metagenomics analysis for presence of specific strain markers will be done in collaboration with the Norwegian node of ELIXIR with researcher Erik Hjerde at the Institute of Chemistry, UiT. The end-users of the knowledge acquired in this project are IBS patients and gastroenterologists as well as the wider scientific community. In Norway, IBS patients are best represented by “”. The board leader of Mage-tarm forbundet, Marion Alver, has kindly agreed to be a user representative. International collaboration is established with Alfonso Benitez Paez at Centro de Investigación Príncipe Felipe, Valencia, Spania whose research into species- and strain-level colonization after FMT will be very beneficial for this project.  The project is funded by Helse-Vest.

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