Bacteria & Inflammation in the Gut (BIG) Study

The null hypothesis is that the clinical response to exclusive enteral nutrition (EEN) and reduction of colonic inflammatory markers are not associated with a characteristic bacterial taxonomy (composition) and metabolites (functionality); and that maintenance of any bacterial changes with supplementary enteral nutrition (SEN), while returning to habitual diet, does not reduce the risk of disease relapse.

The study design will build on previous work by looking at differences in both the gut bacteria (faecal and mucosal) and bacterial metabolic activity at different stages of disease activity. Changes in bacterial composition between disease groups will be examined; between Crohn's disease children on different treatments; and between different sites in the gut. Crohn's disease biomarkers in faeces and urine will also be assessed by analysing bacterial metabolites.

Up to a total of 42 children with Crohn's Disease will be recruited. 42 healthy children will also be recruited to provide a faecal and urine sample, to show the normal variety of gut bacteria and metabolites in healthy children for comparison.

Children who underwent colonoscopy but did not have Crohn's disease will be used as a comparison group to try and identify Crohn's disease bacterial signatures from faeces and urine, and as a non-Crohn's disease control group for comparison of mucosal biopsies.

Recruitment Three groups of children will participate in this study. A. Children undergoing endoscopic investigations (colonoscopy) for colonic inflammation including Crohn's disease [children who are found not to have Crohn's disease will not participate in the follow up aspect (during treatment with EEN) of this study].

B. Previously diagnosed patients with Crohn's disease due to start an 8 week standard course of treatment with EEN due to disease flare up.

C. Healthy children unrelated to Crohn's disease patients will be used as a control group.

Groups A and B: an initial blood, urine, faecal sample followed by 5 further faecal and urine samples over 12 months; and dietary information.

[Group A will also have 8 mucosal biopsies taken during routine endoscopy]. During treatment with EEN (clinical decision), an additional blood sample will be requested during routine blood sampling at start and end of treatment.

Group C: 1 urine and faecal sample will be requested.

All groups will have height, weight and grip strength measured at baseline. Participant receiving EEN will have this repeated 60 days after start of treatment and 60 days after end of treatment.

Detailed Methods Preliminary health check by means of a short health questionnaire - basic health information, age, any medication.

Mucosal samples: in newly diagnosed patients having a colonoscopy for diagnostic purposes, an additional 6 mucosal biopsy samples; 2 from the terminal ileum when possible, 2 from the proximal colon and 2 from the distal colon will be taken (14-16 are collected at normal colonoscopy). An additional 2 biopsy samples will also be collected from the duodenum during upper endoscopy.

Dietary information: habitual dietary intake and eating patterns assessed using validated Food Frequency Questionnaire (FFQ); a 5 step multiple pass 24 hour dietary recall questionnaire for data on actual diet at time of faecal and urine sampling.

EEN compliance (groups A\&B only): completion of short EEN compliance questionnaire at 28 day and 56 day time points.

Medication and Disease History: information on disease activity, medication and medical history collected from medical notes.

Lab Assays Bacterial diversity: The gut bacterial diversity and composition from stool samples will be measured using molecular techniques such as quantitative polymerase chain reaction (PCR) from bacterial ribosomal DNA and next generation sequencing (metagenomics). These techniques will also be used to look at the composition of bacteria associated with mucosal tissue biopsies. Changes in overall gut bacteria composition using standard numerical indices will be measured across the 6 faecal samples collected over the 12 month period, as well as between groups.

Bacterial metabolism: A large number of different products from bacterial metabolism, such as short chain fatty acids, sulphide, and ammonia will be measured using a range of techniques including gas chromatography and gas chromatography mass spectrometry (GCMS) (metabolomics). Faecal pH, a marker of bacterial fermentation, will also be measured.

Disease markers: Other disease markers and metabolites such as faecal calprotectin, will be measured from faecal samples .