The study will investigate the relationship between fecal bile acids, short-chain fatty acids (SCFAs), and the gut microbiota in irritable bowel syndrome (IBS). The central hypothesis of this study is that specific shifts in the GI microbiome composition correlate with altered colonic SCFAs and BAs and contribute to IBS symptoms. Primary aims include: (a) identifying GI microbiome signatures in IBS subtypes (IBS-C and IBS-D) and matched controls, and test if microbiome signatures in these groups correlate with fecal SCFAs and bacterial fermentation of an indigestible carbohydrate (inulin) after a dietary challenge (fecal inulin), and (b) determining if GI microbiome signatures in IBS subtypes and controls correlate with fecal BAs or markers of SCFA production (fecal SCFAs or inulin) and test if BAs correlate with fecal SCFAs or inulin.

The target population is adults ages 18-65 years meeting Rome IV criteria for IBS (both diarrhea- and constipation-predominant, IBS-D and IBS-C) and asymptomatic controls. Primary outcomes will be fecal bile acid excretion and profile, short-chain fatty acid excretion and profile, colonic transit, and fecal microbiota. Secondary outcomes will be stool characteristics based on responses to validated bowel diaries. Stool samples will be collected from participants during the last 2 days of a 4-day 100 g fat diet and split into 3 samples for fecal microbiota, SCFA, and bile acid analysis.

During the study:

Screening/Visit 1

• Participants will be asked to read and sign this informed consent after all questions about the study have been answered to satisfaction.
• This is a screening visit, and we may determine that a participant is ineligible to continue to participate in the study.
• The study team will collect medication and medical history.
• A physical exam will be performed by the physician. Vital signs and body measurements will be collected.
• Participants will be provided with a take-home lasagna meal (vegetarian and non-vegetarian option) and dessert.
• Participants will receive a stool collection kit and instructions for the collection, storage, and transportation of stool.
• Instructions will be provided for a low fiber, high fat diet for Days 2-4.
• Participants will be asked to complete diet recall using the ASA24 and a bowel pattern diary to record bowel symptoms over the course of the study.
• IBS participants will complete the irritable bowel syndrome severity scoring system questionnaires (IBS-SSS).

Day 2-4 (at home):

• Participants will record stool symptoms and patterns in a diary daily.
• Participants will be instructed to consume a low fiber diet (maximum one piece of fruit and 100 g of vegetables a day, and white flour instead of whole grain products), avoid alcohol, and start a 4-day 100 g fat diet.
• On the evening before Day 5, participants will be instructed to collect a stool sample at home and return during Visit 2.

Day 5 (Visit 2):

• Participants will return to the clinic on Day 5 after an overnight fast.
• Participants in the reproductive age range will be given a urine pregnancy test prior to inulin ingestion and the study team will confirm a negative test result.
• Participants will receive breakfast onsite. The non-vegetarian option will consist of pancakes, butter, syrup, hard-boiled eggs, and bacon. The vegetarian option will substitute the bacon with a cheese snack (approximately 790 to 840 kcal) along with 10 g inulin Orafti® powder) thoroughly mixed in 200 mL of water.
• Take-home meals will be provided for lunch and dinner and are to be consumed at 4 and 8 hours from breakfast.
• IBS participants will complete the irritable bowel syndrome severity scoring system (IBS-SSS) questionnaire.
• A stool sample will be collected on Day 5.
• Participants will continue to complete diet recall using the ASA24 and a bowel pattern diary to record bowel symptoms over the course of the study.

Day 6

• Participants will be instructed to collected a final stool sample (Day 6).
• Participants will continue to complete diet recall using the ASA24 and a bowel pattern diary to record bowel symptoms over the course of the study.

Day 7 (Visit 3)

• Participants will return to the clinic to return stool collections (Day 5 and Day 6), the daily diaries, and the IBS-SSS forms (if applicable).

Day 30-90 (Optional dietary follow-up): To further assess the utility of the 24 hour dietary recall, willing participants will be invited to participate in a an optional follow-up activity during which they will complete the Automated Self-Administered Dietary Assessment Tool with or without assistance of a trained interviewer. Interested volunteers will be contacted by phone and provided instructions on how to complete the tool online by the study team.

Inclusion Criteria:

• Age 18-75
• Individuals with irritable bowel syndrome (IBS)
• Healthy volunteers (healthy controls) with no prior history of gastrointestinal (GI) disease or symptoms
• No dietary restrictions other than vegetarian

Exclusion Criteria:

• Inflammatory bowel disease, celiac disease, or certain types of abdominal cancer, as well as those with thyroid or liver issues
• Abdominal surgery or abdominal radiation within 6 months of study participation, with an exception for C-section or gallbladder removal
• Use of any prescription, over the counter, or herbal medications known to affect gastrointestinal function or study interpretation, such as opioids, inflammatory drugs or certain antidepressants, within 6 months prior to study participation for healthy volunteers, or within 2 days of study participation for participants with IBS.
• An exception will be permitted for limited use of stable low doses of antidepressants for individuals who have been taking them for a period greater than one month.
• Rescue medication such as Bisacodyl (dulcolax) to relieve severe constipation and allow for stool collection will be permitted when needed.
• Use of Ozempic and Ozempic-type medications
• Pregnant or breastfeeding women
• Antibiotic use within 3 months of study participation
• Use of prebiotics or probiotics within the 2 weeks before the study initiation
• Regular tobacco use within the past 6 months