Metabolic network restoration, not donor microbial engraftment, associates with resolution of recurrent...

Fecal microbiota transplantation (FMT) effectively prevents recurrent Clostridioides difficile infection (rCDI). However, the mechanisms underpinning FMT efficacy are incompletely understood. We used proximity-ligation metagenomics, metaproteomics, and metabolomics to profile dynamic changes in rCDI. We analyzed samples collected from the first 7 responders during a clinical trial comparing two donor-based treatments (lyophilized FMT [LFMT] or lyophilized sterile fecal filtrate [LSFF], free of intact microorganisms) to prevent rCDI. LSFF is enriched in Caudoviricetes, lipids, fatty acids, and human and extracellular bacterial proteins compared with LFMT. LFMT recipients had donor microbial engraftment, while LSFF recipients achieved only partial microbial community recovery without donor's contribution. Our multi-omics analyses identified a functional convergence following both treatments, shifting away from oxidative and antibiotic stress survival pathways toward enhanced carbon-nitrogen utilization pathways, suggesting that FMT mechanisms may involve metabolic reprogramming and host-microbial amino acid co-metabolism. Our findings provide important hypothesis-generating insights that warrant further validation.