Adaptive evolution of fluoroquinolone resistance in foodborne Salmonella: From metabolic suppression to multidimensional defense mechanisms

The sharp increase of fluoroquinolone (FQ) resistance in Salmonella poses a serious threat to food safety. Elucidating the evolutionary dynamics and mechanisms of FQ resistance is fundamental to its effective prevention and control. Herein, Salmonella Typhimurium was exposed to five FQs for 45 days to investigate resistance evolution, physiological adaptation, and resistance mechanisms. Evolved strains obtained FQ resistance within 3–10 days, with gatifloxacin inducing the highest resistance and fastest cross-resistance, while nalidixic acid was the lowest and slowest. FQ-stressed cells exhibited oxidative stress activation, bioenergetic disruption, biofilm formation, and structural defenses, collectively promoting bacterial adaptation and resistance development. These phenotypic alterations intensified with increasing resistance levels. Genomic analysis of FQ-stressed cells revealed progressive mutations from single gyr A substitution in low-level resistant strains to multiple gyr A/ gyr B and/or par C/ par E mutations in highly resistant bacteria. Structural equation modeling demonstrated biofilm formation as the strongest contributor to FQ resistance, followed by bioenergetic disruption, oxidative stress, and membrane fluidity. Transcriptomic profiling further demonstrated a resistance stage dependent shift of bacterial responses: low-level resistant strains primarily adopted energy conserving metabolic state and iron homeostasis, whereas highly resistant cells developed broader defenses efflux pump activation, reduced outer membrane permeability, enhanced biofilm-associated adaptation, oxidative stress defense, and SOS activation. These findings provide a scientific foundation for deepening the understanding of bacterial resistance evolution, promoting rational antimicrobial use, developing targeted resistance mitigation strategies, and safeguarding food safety and public health.