Evolution of Salmonella Typhimurium under antibiotics from distinct classes reveals variant-dependent cross-protection against food preservation treatments

The growing concern surrounding antimicrobial resistance (AMR) in human bacterial infections has prompted increased investigation into the role of the agri-food sector in AMR transmission and control. While contaminated food is a recognized route of transmission for AMR bacteria, the potential for AMR mechanisms to confer cross-protection and enhance bacterial tolerance to food-relevant preservation stressors such as heat, acidification, or antimicrobial compounds remains poorly understood. In this study, nine resistant variants (RVs) of Salmonella enterica subsp. enterica serovar Typhimurium LT2 were isolated via adaptive laboratory evolution with increasing concentrations of antibiotics from distinct classes, including colistin (COL), amoxicillin (AMX), and erythromycin (ERY). These RVs displayed up to a 16-fold increase in minimum inhibitory concentrations relative to the parental strain. RVs frequently exhibited diverse cross-protection profiles against unrelated antibiotic families. Importantly, certain RVs also showed altered tolerance to food preservation stresses. One COL- and one ERY-RV showed a 10-fold increase in survival under heat treatment (54 °C, 30 min). All ERY-RVs exhibited marked cross-protection to carvacrol (200 μL/L, 30 min), with up to a 1000-fold increase in survival. One COL-RV showed a 100-fold increased tolerance to lactic acid (1% w/v, 40 min). Whole genome sequencing revealed mutations primarily in genes associated with several functional categories, including metabolism and signal processing. Among them, mutations in the efflux regulator ramR and in rfbH, involved in O-antigen synthesis, were identified as candidate determinants potentially linked to cross-protection. The findings suggest that AMR-related mutations in foodborne pathogens such as S. Typhimurium can modify responses to food preservation stresses, highlighting the need to better understand how antibiotic adaptation may influence bacterial persistence under food-related control conditions.