Colonization resistance against Clostridioides difficile is a graded, microbiota-intrinsic property of healthy human gut communities
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Fecal microbiota transplantation cures approximately 90% of recurrent Clostridioides difficile infection, yet it remains unknown whether all healthy donor microbiota confer equivalent protection. We colonized germ-free C57BL/6 mice with stool microbiota from 30 healthy human donors and challenged them with C. difficile in the absence of 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). Of these, 8 conferred Resistant phenotypes, 12 Carrier, 6 mixed Resistant–Carrier outcomes, 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 substantial overlap was observed between groups. Differential abundance analysis identified taxa depleted across non-resistant phenotypes, including Lachnospiraceae taxa such as Hungatella and Sellimonas , and Bacteroides intestinalis . Shotgun metagenomics confirmed these associations and revealed coordinated depletion of biosynthetic and carbohydrate metabolism pathways in non-resistant phenotypes, consistent with broad loss of community metabolic capacity rather than loss of a single dominant function. These findings demonstrate colonization resistance is a graded, microbiota-associated ecological property, evident after host engraftment rather than being 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.
Lay Summary
Clostridioides difficile is a leading cause of serious diarrhea in hospitals. Fecal microbiota transplantation, which transfers stool from a healthy donor to a patient, can cure many recurrent infections. This strongly suggests that healthy gut microbes can protect against this pathogen. However, it is unclear whether all healthy donors provide the same level of protection.
In this study, we colonized germ-free mice with gut microbial communities from thirty healthy adult donors and then challenged them with Clostridioides difficile. The donor communities produced a range of outcomes: some completely blocked the pathogen, some allowed asymptomatic carriage, some caused mild diarrhea, and a few led to severe disease. Importantly, the make-up of donor stool alone did not predict these outcomes. Differences only became apparent after the microbes had established themselves in the mouse gut. Protection was associated with higher overall microbial diversity, certain bacterial groups, and a broad range of metabolic functions rather than any single species.
These findings show that protection is not a fixed property of donor stool, but emerges from how the microbial community establishes and functions in the host. These results have implications for improving donor selection and developing more effective microbiome-based therapies.
