Effects of oxygen and bile acid processing on the small intestinal microbiome and colonization resistance

The small intestine (SI) is a critical site of host-microbe interactions and nutrient absorption, yet it remains severely understudied due to the difficulty of non-invasively sampling its microbiome and environment. Most microbiome studies rely on fecal samples, which primarily reflect the large intestine and fail to capture the distinct microbial ecology of the SI. This knowledge gap limits our understanding of how environmental factors shape microbial composition and function in this essential gut region. Given the SI’s unique chemical landscape—characterized by microaerobic oxygen (O2) levels and high bile acid (BA) concentrations— microbes residing in this niche must adapt to these selective pressures, which likely influence their ability to colonize the SI and contribute to gastrointestinal health and disease. This proposal aims to elucidate how O2 and BAs drive microbial adaptation and interactions within the SI microbiome. We hypothesize that SI-enriched microbes exhibit greater tolerance to O2 and BAs than stool- enriched microbes and that BA metabolism plays a central role in shaping gut ecology by enriching for species capable of processing these antimicrobial compounds. To disentangle the relative contributions of these factors, it is important to isolate and quantify the effects on individual microbes. To do so, we will leverage a novel non-invasive sampling device that enables direct collection of viable SI microbes and metabolites, along with high-throughput culturomics, metagenomics, and metabolomics approaches. Using our established workflows and unique expertise, we will generate diverse isolate libraries and construct in vitro microbial communities that maintain the majority of microbial abundances found in the SI in vivo. We will evaluate the sensitivity of these isolates and communities to a broad range of O2 and BA conditions and determine whether these sensitivities predict the capacity to resist colonization by SI pathogens. These experimental systems will allow us to probe microbial dynamics at scales inaccessible through animal studies and reveal fundamental principles underlying microbial regional preferences in hosts. In Aim 1, we will systematically quantify the effects of O2 and BA concentrations on the growth and metabolism of SI and stool-derived isolates to establish how these factors drive microbial localization within the gut. In Aim 2, we will investigate the emergent effects of BA metabolism and O2 tolerance in microbial communities. Using in vitro community models, we will probe how these environmental factors modulate microbiome structure and function, particularly in the context of colonization resistance against pathogens linked to diseases such as irritable bowel syndrome (IBS) and small intestinal bacterial overgrowth (SIBO). This work will bridge long- standing gaps in microbiome science by providing novel insights into SI microbial ecology and a mechanistic framework for designing microbiome-based interventions, including personalized probiotics and targeted therapies for SI-associated diseases. Project Number: 1R21AI197001-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: KERWYN HUANG | Institution: STANFORD UNIVERSITY, STANFORD, CA | Award Amount: $423,500 | Activity Code: R21 | Study Section: Interspecies Microbial Interactions and Infections Study Section[IMII] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19700101