Exposure Regimen and Selecting Antibiotic Determine Resistance and Growth Dynamics of Ampicillin- and Gentamicin-Evolved Salmonella Typhimurium

Antimicrobial resistance (AMR) may alter bacterial traits beyond antibiotic susceptibility, but its physiological consequences remain unclear. This study investigated whether these outcomes in Salmonella Typhimurium depend on the antibiotic, ampicillin (AMP) or gentamicin (GEN), and the selection regimen. Two adaptive laboratory evolution (ALE) strategies were compared: constant subinhibitory exposure (Assay A) and stepwise increasing antibiotic exposure (Assay B). Minimum inhibitory concentrations, whole-genome sequencing, cross-resistance profiles, and growth kinetics were evaluated across at least three biological replicates. Assay A produced resistance increases up to 8-fold, with only one variant reaching the resistance breakpoint, whereas Assay B yielded increases up to 128-fold for AMP-resistant variants (RVs) and 16-fold for GEN-RVs. AMP resistance was associated with mutations in acrB and ramR, and broader cross-resistance, with generally unchanged lag phases but strain-dependent reductions in maximum growth rates. In contrast, GEN resistance involved mutations related to respiration and energy production (cydA, hemL, etc.), limited cross-resistance and growth impairment across GEN-RVs, including small-colony variant (SCV)-like phenotypes. In an exploratory assay, one GEN-RV showed delayed plaque expansion during Dictyostelium discoideum predation, suggesting altered interactions with phagocytic predators. Overall, AMR-associated phenotypes depend on both antibiotic and selection regimen, underscoring the importance of physiological changes when assessing resistant foodborne pathogens.