Original Investigation of CRISPR-Cas System Diversity and Its Role in Horizontal Gene Transfer Suppression in Clinical Gram-Negative Pathogens Across Nigerian Tertiary Hospitals

CRISPR-Cas systems in bacteria function as adaptive immune mechanisms against horizontal gene transfer (HGT), including the acquisition of antibiotic resistance plasmids, yet CRISPR diversity and its influence on resistance gene acquisition in Nigerian clinical gram-negative pathogens constitute an entirely unexplored research domain. This dissertation investigates CRISPR-Cas system diversity and its relationship to horizontal resistance gene transfer in clinical gram-negative pathogens across tertiary hospitals in Nigeria, making original contributions to molecular microbiology and AMR evolutionary biology. Clinical isolates of Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa from hospitals in Sokoto (North West), Anambra (South East), Edo (South South), and Kwara (North Central) were whole-genome sequenced. CRISPR array detection and Cas protein typing used CRISPRFinder and CRISPRCasTyper. Resistance gene carriage was determined using ResFinder. Conjugation frequency assays quantified plasmid acquisition rates in CRISPR-positive versus CRISPR-negative strains. CRISPR-Cas systems were identified in 38 percent of K. pneumoniae, 29 percent of E. coli, and 44 percent of P. aeruginosa isolates. CRISPR-positive strains carried significantly fewer acquired resistance genes per isolate (mean 2.1) compared to CRISPR-negative strains (mean 4.8, p < 0.001). Type I-E systems predominated in K. pneumoniae, while Type I-F dominated in P. aeruginosa. Spacer sequence analysis confirmed targeting of resistance plasmid protospacers in functional CRISPR arrays. The dissertation provides the first characterisation of clinical gram-negative CRISPR landscapes from Nigeria. Keywords: CRISPR-Cas, horizontal gene transfer, antimicrobial resistance, gram-negative pathogens, Nigeria