{"paper_id":"40ccd8e3-e8cd-48ae-94ec-5834338ce0e1","body_text":"Abstract\nBackground Invasive non-typhoidal Salmonella (iNTS) disease remains a major public health challenge in sub-Saharan Africa. Salmonella Typhimurium is responsible for the majority of cases, with specific lineages being associated with increased risk of bloodstream infection. We have recently developed a Salmonella Typhimurium controlled human infection model (CHIM) to better understand disease pathogenesis and to provide a platform to test candidate vaccines. Selecting appropriate challenge strainsis a central design consideration in developing challenge model protocols. We describe the rationale, manufacture, and detailed characterisation of the two Salmonella Typhimurium strains used in the first-in-human NTS CHIM.\nMethods Two Salmonella Typhimurium strains, 4/74 (ST19) and D23580 (ST313), were selected from the UK Health Security Agency National Collection of Type Cultures and manufactured under Good Manufacturing Practice (GMP) conditions. Challenge agent stocks underwent microbial limits testing, viability and stability assessments, and phenotypic characterisation including growth kinetics, motility, acid sensitivity, and antibiotic susceptibility. Whole-genome sequencing was performed to confirm genetic stability post-manufacture. The effect of pre-challenge handling conditions was assessed in saline and sodium bicarbonate buffers, and bacterial survival was evaluated under simulated gastric and intestinal conditions using a modified Rossett–Rice model. Transcriptomic profiling was undertaken to determine whether sodium bicarbonate exposure altered expression of key virulence genes.\nResults Both strains retained their expected phenotypic characteristics, including reduced motility and melibiose utilisation in D23580. GMP stocks remained pure, viable, and stable post-manufacture, with no unexpected genomic mutations detected. Both strains were susceptible to clinically relevant antibiotics used in the study. Survival was maintained in neutral and mildly acidic conditions, with significant reduction below pH 3.5. Stability was preserved for up to one hour in saline buffer and during simulated gastric transit in the Rossett-Rice model. Transcriptomic analysis showed no significant changes in Salmonella pathogenicity island-1 or 2, or flagellar gene expression following sodium bicarbonate exposure\nConclusions These findings confirm that the Salmonella Typhimurium 4/74 and D23580 strains retained genomic and phenotypic integrity post-manufacture. The resulting challenge stocks provide the foundation for an ongoing NTS CHIM that aims to advance understanding of NTS and iNTS pathogenesis and support candidate vaccine testing.\nCompeting Interest Statement\nThe authors have declared no competing interest.\nFootnotes\nIn this revised manuscript, we have expanded the Methods section to include additional procedural detail on GMP manufacture, microbial limits testing, the Rossett-Rice model, and RNA extraction and sequencing. The Transcriptomics section has been updated with further description of the RNA-seq analysis and confirmation of data deposition in the NCBI Gene Expression Omnibus (GEO). Figures have also been reformatted into multi-panel layouts to enhance clarity and presentation of the data.","source_license":"CC-BY-4.0","license_restricted":false}