A Systems Biology Approach to Understanding Antibiotic Tolerance and Heteroresistance in Staphylococcus aureus Clinical Isolates from Neonatal Units in Nigeria

Antibiotic tolerance and heteroresistance in Staphylococcus aureus, phenomena distinct from stable acquired resistance, enable bacterial survival during antibiotic treatment and predispose to therapeutic failure in neonatal infections, yet their prevalence and mechanistic basis in Nigerian neonatal unit clinical isolates have not been investigated. This dissertation applies a systems biology approach to characterise antibiotic tolerance and heteroresistance in Staphylococcus aureus from neonatal units at five teaching hospitals spanning Kaduna (North West), Imo (South East), Ekiti (South West), Kogi (North Central), and Rivers (South South) States. Staphylococcus aureus isolates were screened for tolerance by minimum duration of killing assays and heteroresistance by population analysis profiling against vancomycin, oxacillin, and daptomycin. Tolerant and heteroresistant isolates underwent whole transcriptome RNA-seq under antibiotic challenge conditions. Metabolomics using GC-MS profiled metabolic shifts accompanying tolerance. Regulatory network modelling using a constraint-based metabolic reconstruction identified candidate tolerance gene targets. Tolerance was confirmed in 31 percent of isolates and heteroresistance in 19 percent, with overlap in 12 percent. RNA-seq identified upregulation of stringent response (relA, spoT), cell wall remodelling (pbpX), and reactive oxygen species detoxification pathways in tolerant isolates. Metabolomics confirmed tricarboxylic acid cycle downregulation and alternative fermentation pathway activation as metabolic tolerance signatures. Regulatory network modelling identified the stringent response as a high-priority therapeutic targeting node. Keywords: antibiotic tolerance, Staphylococcus aureus, neonatal unit, systems biology, Nigeria