Bloodstream-associated Salmonella Typhimurium and Enteritidis iNTS pathovariants hyper-replicate in human macrophages

Background Invasive non-typhoidal Salmonella (iNTS) are a major cause of bloodstream infections in sub-Saharan Africa, yet the mechanisms that allow iNTS bacteria to survive within human cells remain poorly understood. Macrophages represent a key intracellular niche for Salmonella during systemic infection, and a reproducible human model for iNTS is required to compare pathovariants behaviour. Methods A human macrophage infection model was developed and optimized based on PMA-differentiated THP-1 cells to investigate infection dynamics of clinically relevant Salmonella Typhimurium and Salmonella Enteritidis strains. We compared intracellular survival and replication of gastroenteritis-associated and bloodstream-associated pathovariants, including S. Typhimurium ST313 Lineage 2, the novel S. Typhimurium ST313 Lineage 3 and the understudied S. Enteritidis Central/Eastern African (CEAC) clades. Bacterial uptake and intracellular replication were distinguished by gentamicin protection assay and confocal microscopy. Macrophage activation state was assessed to determine the capacity of the model to reproduce pro-inflammatory polarisation and macrophage plasticity. Results CEAC S. Enteritidis and ST313 S. Typhimurium iNTS pathovariants hyper-replicate within human macrophages compared to global epidemic isolates, identifying a shared intracellular phenotype. The cellular model achieved robust pro-inflammatory polarisation of human macrophages, while revealing limitations in modelling macrophage plasticity. Conclusions Intracellular behaviour differs between Salmonella pathovariants, and enhanced replication within human macrophages is shared by African iNTS lineages of both serovars, Typhimurium and Enteritidis. The optimised THP-1 model provides a reproducible framework for dissecting invasive disease pathogenesis and evaluating therapeutic strategies against iNTS bacteria.