Successful Fecal Microbiota Transplants in Post-antibiotic Treated Recurrent Clostridioides difficile Patients Induce Acylcarnitine and Sphingolipid Lipidomic Shifts

Clostridioides difficile infection (CDI) is an urgent public health threat in the United States, resulting on an annual basis in over half a million cases, more than 29,000 deaths, and $4.8 billion in healthcare costs. While fecal microbiota transplants (FMTs) have proven more effective than standard-of-care antibiotics in resolving recurrent CDI (rCDI), their inherent risks underscore the need for advancements in regulated alternative therapies such as live biotherapeutic products (LBPs). The development of effective LBPs, however, is contingent upon better understanding the biological mechanisms underlying FMT efficacy. Building on our previously published untargeted metabolomic study which identified lipids as major explanatory factors associated with successful FMTs, we assessed additional lipid species using an instrumental platform coupling liquid chromatography, ion mobility spectrometry, collision induced dissociation, and mass spectrometry (LC-IMS-CID-MS) techniques. This platform and data analysis workflow enable the evaluation of >850 unique lipid species across 26 classes. Here, we confidently identified 397 lipids in the stools of 15 rCDI patients at pre- and post-FMT (2 week, 2 month, and 6 month) time points. Statistical evaluations of the lipidomic data illustrated that FMT-administration drastically reshapes the lipidome (adonis test, R ² =0.11999, Pr(>F) <0.001), including 96 specific lipid species across 18 lipid classes (mixed effects modeling, BH correction, p < 0.05). In particular, we noted that medium and long-chain acylcarnitines decreased following FMT administration, while very long-chain acylcarnitines were elevated in post-FMT samples. Additionally, we observed assayed sphingolipids to be elevated pre-FMT with the exception of trihydroxy ceramides, which were highly upregulated post-FMT. These lipidomic alterations suggest that FMT administration may influence intestinal barrier integrity, inflammatory signaling, or apoptosis pathways. Interestingly, there was a strong co-occurrence of medium and long-chain acylcarnitines with Enterobacteriaceae, a bacterial family that has been demonstrated to utilize carnitine for growth. These findings highlight the critical role of the lipidome in patient susceptibility to rCDI and suggest the interactions between microbiota and lipids pre- and post-FMT as targets for developing next-generation LBPs.