Epidemiological and Evolutionary Genomics of Clostridioides difficile in Healthcare Settings Across Multiple Geopolitical Zones of Nigeria: A Multi-Centre Longitudinal Study

Clostridioides difficile infection (CDI) is the foremost antibiotic-associated nosocomial diarrhoeal disease globally, yet its burden, genomic diversity, and evolutionary trajectory in Nigeria remain entirely uncharacterized at a national scale. This multi-centre longitudinal study established the epidemiological and evolutionary genomics of C. difficile in healthcare settings across the six geopolitical zones of Nigeria. Toxigenic C. difficile isolates were prospectively collected from diarrhoeal patients meeting CDI case definitions at 12 sentinel hospitals over 30 months. Whole-genome sequencing of all 180 recovered toxigenic isolates was performed. Multi-locus sequence typing, core-genome phylogeny, toxin gene characterization (tcdA, tcdB, cdtA/cdtB, tcdC), and antimicrobial resistance determinant analysis were conducted. A pan-genomic analysis of all Nigerian isolates relative to global reference databases identified Nigeria-specific genetic features. Bayesian phylogeographic analysis traced national transmission routes and dated key evolutionary events. Results showed a CDI prevalence of 13.7 percent across enrolled diarrhoeal patients. Ribotype 078 was the dominant lineage (38.3%), followed by RT002 (21.1%) and RT017 (16.7%). Binary toxin genes were detected in 44 percent of isolates. A novel emerging clade, provisionally designated Nigeria Clade 1 (NgC1), was identified in 14 isolates from the South East and South South zones and exhibited a unique toxin gene arrangement not previously reported in global databases. Metronidazole resistance was absent, but moxifloxacin resistance was detected in 62 percent of RT078 isolates. These findings constitute an original epidemiological and genomic contribution to CDI science in Africa and establish the first nationally representative Nigerian C. difficile genome database. Keywords: Clostridioides difficile, evolutionary genomics, ribotyping, CDI epidemiology, Nigeria.