Valappil, Vishnu Thayyil

Abstract The conventional bottom-up approach to probing the human gut microbiome’s link with hosts in germ-free models is hampered by considerable costs and time. To address this, our study introduces the nematode Caenorhabditis elegans as an innovative high-throughput model for exploring the gut microbiome’s impact on functional phenotypes. Traditionally, C. elegans studies have used continuous feeding for bacterial administration, a method that is unsuitable for anaerobes. For the first time, we have standardized a protocol for colonizing C. elegans with human gut anaerobes. By screening a microbial culturomics library representing 70% of the gut microbiome’s functional capacity, we showed successful colonization for 46% of the library. Functional phenotyping revealed that 5 of 10 strains, previously identified in vitro as inhibiting C. difficile , also inhibited in vivo . Validation of a selected strain in a germ-free mouse model confirmed colonization resistance and an immune response consistent with findings in C. elegans , underscoring the model’s translational potential.