A Functional Genomics Investigation of Sporulation and Germination Regulatory Networks in Clostridioides difficile Strains Isolated from Nigerian Healthcare Settings

Clostridioides difficile infection (CDI) is an emerging healthcare threat in Nigerian hospitals, driven by spore persistence, antibiotic disruption of gut flora, and patient-to-patient transmission, yet functional genomic characterisation of C. difficile sporulation and germination regulatory networks in Nigerian clinical strains has not been conducted. This dissertation employs functional genomics to investigate sporulation and germination regulation in C. difficile isolates from Nigerian healthcare settings, contributing original mechanistic understanding of Nigerian clinical C. difficile biology. Clinical C. difficile isolates were recovered from stool samples of diarrhoeal patients at teaching hospitals in Enugu (South East), Abuja (North Central), and Lagos (South West). Whole genome sequencing and multilocus sequence typing characterised isolate diversity. RNA-seq transcriptomic profiling under sporulation-inducing and vegetative growth conditions identified differentially expressed sporulation and germination regulatory genes. Spo0A, sigE, sigK, gerA, and gerB expression kinetics were characterised by RT-qPCR. Selected regulatory gene knockout mutants were constructed using TargeTron technology and phenotypically evaluated for sporulation frequency and germination efficiency. Nigerian clinical isolates exhibited enhanced spo0A expression and elevated sporulation frequencies compared to reference strain 630, correlating with prolonged environmental persistence in simulated hospital surface conditions. Spo0A overexpression correlated with hospital cleaning agent tolerance. The dissertation provides original functional genomics data on Nigerian C. difficile clinical biology and identifies spo0A as a potential therapeutic target for anti-sporulation intervention strategies. Keywords: Clostridioides difficile, sporulation, functional genomics, Nigeria, healthcare infection