Untargeted metabolomics identifies faecal filtrate-derived metabolites that disrupt Clostridioides difficile metabolism and confer gut barrier cytoprotection

Qassadi, FI, Johnson, C, Robinson, K, Griffin, R, Polytarchou, C ORCID: https://orcid.org/0000-0002-1948-7934, Kao, D, Kim, D-H, Griffiths, RL, Zhu, Z and Monaghan, TM, 2025. Untargeted metabolomics identifies faecal filtrate-derived metabolites that disrupt Clostridioides difficile metabolism and confer gut barrier cytoprotection. International Journal of Molecular Sciences, 26 (22): 11221. ISSN 1661-6596

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Abstract

Recurrent Clostridioides difficile infection (rCDI) remains a major therapeutic challenge. Although faecal microbiota transplantation (FMT) is highly effective and thought to restore microbial composition and metabolic function, the mechanisms underlying its success are not fully understood. In particular, the contribution of non-bacterial components such as soluble metabolites remains unclear. Therefore, further investigation is needed to identify the mechanistic drivers of FMT efficacy and clarify how non-bacterial factors contribute to therapeutic outcomes. Here, we applied untargeted three-dimensional Orbitrap secondary ion mass spectrometry (3D OrbiSIMS) to profile faecal metabolic reprogramming in rCDI patients pre- and post-FMT, alongside C. difficile cultures exposed to sterile faecal filtrates. FMT induced extensive metabolic shifts, restoring glyoxylate/dicarboxylate and glycerophosphoinositol pathways and normalising disrupted bile acid and amino acid profiles. Faecal filtrate exposure caused strain-specific metabolic disruption in C. difficile, depleting proline, fumarate and succinate while enriching tryptophan. While multiple metabolite classes were profiled, the most significant functional changes were observed in lipids. Lipidomics identified >3.8-fold enrichment of phosphatidylinositol (PI) species, which localised to bacterial membranes and conferred cytoprotection against C. difficile toxins and other epithelial insults. Spatial metabolomics imaging revealed, for the first time, metabolite compartmentalisation within C. difficile, with proline and succinate broadly distributed across the cell surface and fumarate confined to distinct microdomains, highlighting functional heterogeneity in pathogen metabolism. Collectively, these findings demonstrate that soluble metabolites within faecal filtrates mediate pathogen suppression and epithelial barrier protection, establishing metabolite-driven mechanisms underlying FMT efficacy and identifying PI lipids as candidate post-biotic therapeutics for rCDI.

Item Type:	Journal article
Publication Title:	International Journal of Molecular Sciences
Creators:	Qassadi, F.I., Johnson, C., Robinson, K., Griffin, R., Polytarchou, C., Kao, D., Kim, D.-H., Griffiths, R.L., Zhu, Z. and Monaghan, T.M.
Publisher:	MDPI
Date:	20 November 2025
Volume:	26
Number:	22
ISSN:	1661-6596
Identifiers:	Number Type 10.3390/ijms262211221 DOI 2565440 Other
Rights:	© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Divisions:	Schools > School of Science and Technology
Record created by:	Jonathan Gallacher
Date Added:	03 Feb 2026 12:42
Last Modified:	03 Feb 2026 12:43
URI:	https://irep.ntu.ac.uk/id/eprint/55171

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