Abstract / Summary
Abstract The success of fecal microbiota transplantation and live biotherapeutic products in preventing recurrent Clostridioides difficile infection demonstrates that healthy gut microbiota can confer colonization resistance, yet it remains unknown whether all healthy donor microbiota confer equivalent protection. We colonized germ-free C57BL/6 mice with stool microbiota from 29 healthy human donors and challenged them with C. difficile without antibiotic pretreatment. Donor microbiota conferred a spectrum of colonization resistance phenotypes: Resistant (no detectable colonization or toxin), Carrier (asymptomatic colonization with detectable toxin), Symptomatic (non-lethal diarrheal illness), and Susceptible (lethal infection). Eight donors conferred Resistant, 11 Carrier, 6 mixed Resistant–Carrier, and 4 Symptomatic or Susceptible phenotypes. While 16S rRNA gene sequencing of donor stool did not distinguish phenotypes across any diversity or compositional metric tested, humanized mouse microbiomes exhibited clear phenotype-dependent differences after engraftment. Richness (observed amplicon sequence variants) and diversity (Shannon and Faith’s phylogenetic diversity) declined progressively from Resistant to Susceptible phenotypes, although groups overlapped substantially. Differential abundance analysis identified taxa depleted across non-resistant phenotypes, including Lachnospiraceae such as Hungatella and Sellimonas, and Bacteroides intestinalis. Shotgun metagenomics confirmed these associations and revealed coordinated depletion of biosynthetic and carbohydrate metabolism pathways in susceptible microbiome, consistent with broad loss of community metabolic capacity rather than of a single dominant function. These findings demonstrate colonization resistance is a graded, microbiota-associated ecological property, evident after host engraftment rather than a binary trait encoded in donor stool. This has implications for donor screening in fecal microbiota transplantation and the rational design of microbiome-based therapeutics.