Long-Read Nanopore Sequencing-Based Complete Characterisation of Plasmid-Borne Resistance in Priority Gram-Negative Nosocomial Pathogens Across Nigerian Tertiary Hospitals

Plasmid-mediated resistance in Gram-negative nosocomial pathogens is driven by complex, incompletely characterised mobile genetic elements that short-read sequencing approaches cannot resolve, leaving a critical knowledge gap in understanding resistance dissemination across Nigerian hospitals. This research employs Oxford Nanopore Technologies long-read sequencing to achieve complete plasmid characterisation of carbapenem and colistin-resistant Gram-negative pathogens from tertiary hospitals across four geopolitical zones. Carbapenem-resistant Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates meeting predefined resistance criteria will be collected from hospitals in Lagos, Kano, Enugu, and Bauchi States over eighteen months. Complete closed genomes will be assembled using Flye, with polishing by Medaka. Plasmid replicon typing, relaxase typing, and synteny analysis will characterise plasmid diversity and evolutionary relationships. Identification of integrative conjugative elements, insertion sequences, and transposons will elucidate mobilisation mechanisms. Pan-genome analysis will reveal resistance gene distribution patterns across hospitals and species. Transfer competency will be experimentally confirmed by conjugation assays. Phylogenomic reconstruction will determine inter-hospital and inter-species plasmid transmission routes. Original contributions include the most comprehensive complete plasmid resistome characterisation of Nigerian nosocomial pathogens, a theoretical model of resistance plasmid evolution under Nigerian hospital selection pressures, and identification of high-risk plasmid types warranting targeted infection prevention responses. Findings will directly inform the National AMR Action Plan genomic surveillance strategy. Keywords: Nanopore sequencing, plasmid characterisation, carbapenem resistance, nosocomial pathogens, mobile genetic elements