Metatranscriptomic signatures of recurrent Clostridioides difficile infection reveal functional dysbiosis driving relapse | ||
| Microbes and Infectious Diseases | ||
| Articles in Press, Accepted Manuscript, Available Online from 13 November 2025 | ||
| Document Type: Original Article | ||
| DOI: 10.21608/mid.2025.415743.3133 | ||
| Authors | ||
| Ibrahim Ismael Shahad1; Rafal Ismael Ali2; Ahmed Arnaoty* 1, 3 | ||
| 1Bilad Alrafidain University, College of health and medical techniques, Iraq | ||
| 2Tropical-Biological Research Unit, College of Science, University of Baghdad, Baghdad, Iraq | ||
| 3Ibn Sina University, College of medicine, Iraq | ||
| Abstract | ||
| Background: Relapse is common in Clostridioides difficile infection (CDI) and remains difficult to prevent, partly because standard DNA-based profiling misses real-time microbial activity during recurrence. Aim: To quantify, in a prospective Iraqi cohort (n=12; January–October 2024), how community functional activity and C. difficile virulence transcription change across baseline, active infection, and relapse, and to evaluate a simple relapse-risk model based on combined host–microbiota features. Methods: Serial stool samples underwent rRNA-depleted RNA sequencing and downstream community/pathway profiling, with targeted assessment of C. difficile virulence and sporulation transcripts. Network correlations linked pathogen transcripts to community metabolic pathways. A multivariable logistic model explored relapse-risk prediction. Results: Eight of twelve patients (67 %) experienced relapse within 18–35 days (median 27 days). Shannon diversity declined from 3.6 ± 0.4 at baseline to 2.1 ± 0.3 during infection and remained suppressed at relapse (p < 0.01). Relapse samples showed expansion of Clostridium XI and Enterococcus with depletion of Faecalibacterium and Bacteroides. Functionally, Stickland amino-acid fermentation increased p = 0.001), whereas butyrate biosynthesis ( p = 0.004) and bile-acid 7α-dehydroxylation p = 0.002) were transcriptionally repressed. C. difficile virulence genes were upregulated: tcdA, tcdB and spo0A. A multivariable model incorporating tcdA expression, Stickland:butyrate pathway ratio, and baseline Faecalibacterium achieved moderate discrimination (AUC = 0.68, 95 %). Conclusions: CDI relapse reflects functional dysbiosis coupled to virulence activation, rather than pathogen persistence alone. Metatranscriptomics identifies actionable biomarker candidates and supports microbiota-targeted strategies (e.g. butyrogenic and bile-acid–restoring approaches) to reduce relapse risk in resource-limited settings. | ||
| Keywords | ||
| Clostridioides difficile Infection (CDI); Microbiota; Dysbiosis; Metatranscriptomics | ||
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