Fecal bile acid profiles predict recurrence in patients with primary Clostridioides difficile infection

Background Factors that influence recurrence risk in primary Clostridioides difficile infection (CDI) are poorly understood, and tools to predict recurrence are lacking. Perturbations in microbial-derived bile acids (BAs) contribute to CDI pathogenesis and may be relevant to primary disease prognosis. Aims To define stool bile acid profiles and microbial bile-metabolising functionality in primary CDI patients, and explore signatures predicting recurrence. Methods Weekly stool samples were collected from primary CDI patients from the last day of anti-CDI therapy until recurrence, or through eight weeks post-completion otherwise. Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was used to profile bile acids, and bacterial bile salt hydrolase (BSH) activity was measured to determine primary BA deconjugation capacity. Multivariate and univariate models were used to define differential BA trajectories in recurrers versus non-recurrers, and assess fecal bile acids as predictive markers for recurrence. Results Twenty (36%) out of 56 patients (median age 57, 64% male) recurred, with 80% of recurrence occurring within the first nine days post-antibiotic treatment. Principal component analysis (PCA) of stool bile acid profiles demonstrated clustering of samples by recurrence status and post-treatment time point. Longitudinal fecal bile acid trajectories in non-recurrers showed a recovery of secondary bile acids and their derivatives in non-recurring patients that was not observed in recurrers. BSH activity increased over time amongst patients who did not relapse (β= 0.056; likelihood ratio test p =0.018). A joint longitudinal-survival model identified five stool bile acids with AUROC > 0.73 for prediction of recurrence within nine days post-CDI treatment. Conclusions Gut bile acid metabolism dynamics differ in primary CDI patients between those who develop recurrence versus those who do not. Individual bile acids show promise in primary CDI patients as potential novel biomarkers to predict CDI recurrence. ### Competing Interest Statement BHM has received consultancy fees from Finch Therapeutics Group and Ferring Pharmaceuticals, outside of the submitted work. JRFW has received consultancy fees from GE Healthcare, Intercept Pharmaceuticals, and Novome Biotechnologies outside the submitted work. JRM has received consultancy fees from EnteroBiotix Ltd., outside of the submitted work. JRA has consulted for Finch Therapeutics Group, Seres, Pfizer, Janssen, Bristol Myer Squibb, Artugen, Iterative Scopes, Lilly, Merck and Abbvie, and has research support from Merck and Pfizer. ### Funding Statement The clinical studies were funded by a Junior Faculty Development Award from the American College of Gastroenterology for JRA. Metabolomics studies were performed at the MRC-NIHR National Phenome Centre at Imperial College London; this centre receives financial support from the Medical Research Council (MRC) and National Institute of Health Research (NIHR) (grant number MC\_PC\_12025). BHM is the recipient of an NIHR Academic Clinical Lectureship (CL-2019-21-002) and was formerly in receipt of a MRC Clinical Research Training Fellowship (MR/R000875/1). The Division of Digestive Diseases and MRC-NIHR National Phenome Centre at Imperial College London receive financial and infrastructure support from the NIHR Imperial Biomedical Research Centre (BRC) based at Imperial College Healthcare NHS Trust and Imperial College London. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Institutional Review Board (IRB) of Brigham and Womens Hospital gave ethical approval for this work. In addition, a UK National Research Ethics Center (13/LO/1867) also gave ethical approval for this. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The bile acid profiling data generated for this project is available on the MetaboLights Repository, study number: MTBLS657. All data produced in the present study are available upon reasonable request to the authors.