The impact of low-abundance commensals on gut colonization resistance

Clostridioides difficile infection (CDI) poses a significant public health burden, with approximately half a million cases annually in the United States and recurrence rates of up to 25%. CDI is primarily driven by disruptions to the gut microbiome, often due to antibiotic treatment, which creates an environment conducive to C. difficile (Cd) colonization. Current microbiome-based therapeutic strategies, such as fecal microbiota transplants (FMTs), have shown promise in restoring colonization resistance; however, their safety, reproducibility, and regulatory challenges limit widespread clinical application. While defined microbial consortia represent a promising alternative, their efficacy remains suboptimal. We hypothesize that these failures stem at least in part from an over-reliance on high-abundance (HA) species and an incomplete understanding of how low- abundance (LA) species contribute to microbiome stability and pathogen resistance. This project seeks to systematically evaluate the role of LA species in microbiome assembly and in conferring colonization resistance using a combination of synthetic microbial communities, metabolomics, and gnotobiotic mouse models. We hypothesize that LA species play crucial metabolic and ecological roles, through direct competition with Cd and by reinforcing community resilience under antibiotic-induced perturbations. To test this hypothesis, we will employ a defined yet complex synthetic community, mhCom, that encompasses both HA and LA species and assembles reproducibly in vitro and in vivo. Leveraging high-resolution metabolomics, we will (i) characterize the metabolic niches and functional redundancies of LA species and (ii) determine their role in resistance to Cd colonization and microbiome recovery and Cd suppression following antibiotic-induced CDI. In Aim 1, we will map the metabolic functions of LA species within mhCom in vitro, identifying privileged metabolic niches and cross-feeding interactions that contribute to community stability. We will use untargeted metabolomics and species dropouts to establish whether LA species and/or Cd fill metabolic voids when HA species are lost. In Aim 2, we will use gnotobiotic mice to assess the impact of LA species on C. difficile colonization resistance, determining whether the inclusion of LA species enhances pathogen exclusion both before and after antibiotic-induced microbiome disruption. By addressing a critical knowledge gap in microbiome ecology, this study has the potential to redefine microbiome-based therapeutics, either by demonstrating that LA species are not merely passive members of the gut community but essential contributors to microbiome resilience or by affirming strategies focused on HA species. The outcomes of this research will inform the rational design of next-generation microbial therapeutics with enhanced robustness against CDI and will have direct impact for other microbiome-related diseases, providing a foundation for safer, more effective, and precision-targeted microbiome interventions. Project Number: 1R21AI196913-01 | 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: $434,090 | Activity Code: R21 | Study Section: Interspecies Microbial Interactions and Infections Study Section[IMII] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI19691301