Antibiotic-Driven Evolutionary Engineering in Salmonella Heidelberg Reveals Genomic Signatures of Attenuation

<b>Background/Objectives</b>: Salmonella Heidelberg (SH) is a globally distributed pathogen associated with gastrointestinal disease in humans and animals and frequently affects poultry. Among the classic strategies used in vaccine development, evolutionary engineering enables the generation of attenuated bacterial strains through exposure to selective pressures such as antibiotics. In this study, spontaneous antibiotic-resistant mutant strains of SH were generated by exposure to high concentrations of streptomycin and rifampicin, after which their phenotypic and genotypic characteristics were evaluated. <b>Methods</b>: The wild-type strain SA628 <i>wt</i> was subjected to continuous and discontinuous selection under antibiotic pressure. Phenotypic characterization included biochemical profiling and antibiotic susceptibility testing. Whole-genome sequencing was performed to identify genetic changes affecting virulence- and resistance-associated genes, plasmid content, and point mutations using variant calling approaches. The potential functional relationships of the mutated genes were further analyzed through genetic network analysis. <b>Results</b>: The mutant strains SA628 <i>mut1</i> and SA628 <i>mut3</i> were obtained through discontinuous selection, whereas strain SA628 <i>mut2</i> was generated under continuous selection. Phenotypically, all the mutant strains exhibited resistance to streptomycin, whereas SA628 <i>mut2</i> and SA628 <i>mut3</i> also exhibited resistance to rifampicin. Genomic analyses revealed mutations in <i>rpoS</i>, <i>ascD</i>, <i>ynfE</i>, <i>rpoB</i>, and <i>cyaA</i> associated with discontinuous selection and in <i>iscU</i>, <i>ybiO</i>, <i>rpoB</i>, and <i>rsmG</i> associated with continuous selection. Network analysis indicated that these genes are functionally connected within regulatory and metabolic interaction networks, including global transcriptional regulation, anaerobic metabolism, cAMP-mediated signaling, translation, and iron-sulfur cluster biogenesis. <b>Conclusions</b>: Collectively, these findings suggest that antibiotic-driven selection promotes coordinated genetic changes affecting stress responses and metabolism, which may contribute to reduced virulence. This work provides insights into bacterial adaptation under antibiotic stress and supports the potential use of evolutionary engineering for the development of attenuated strains.