Ciprofloxacin-resistant variants of Listeria monocytogenes EGD-e show increased heat resistance in buffer and milk

• Evolved resistant variants of L. monocytogenes EGD-e emerged under antibiotic stress • Ciprofloxacin resistant variants displayed cross-protection to heat in skimmed milk • Mutations in genes related to antibiotic target and efflux pumps are involved • Mutation in stress response genes seems to be related to oxytetracycline resistance • Mutations in cell wall-related genes are suggested to increase thermoresistance The extensive use of antibiotics in primary production has promoted the emergence of resistant bacteria. Due to cross-protection phenomena, these antimicrobial resistant (AMR) bacteria may also withstand food preservation treatments applied in the food industry. This study aimed to evaluate the emergence of resistant variants (RVs) of Listeria monocytogenes EGD-e after prolonged exposure to antibiotics (amoxicillin, ciprofloxacin and oxytetracycline) based on adaptive laboratory evolution assays. RVs were selected by determining the minimum inhibitory concentration, then characterized phenotypically against heat treatments (58 °C/ 20 min) and genotypically to identify mutations responsible for changes in thermoresistance. Five ciprofloxacin RVs (Lm Cip1-5 ) and one oxytetracycline RV (Lm Oxy ) were obtained. Several ciprofloxacin RVs showed greater thermoresistance in McIlvaine buffer (pH 7.0) than the parental strain, also observed in skimmed milk (pH 6.8). Mutations identified in codY (Lm Oxy ) and fepR and parC (ciprofloxacin RVs) are likely responsible for the antibiotic resistance. Moreover, mutations in genes linked to cell wall biosynthesis ( rml ), metabolism and RNA or energy processing (e.g., cshA, atpA2, lmo2794 ) may contribute to increased thermoresistance. These findings highlight the interaction between AMR and cross-protections mechanisms, and the potential risk posed by AMR bacteria in the food chain, which could compromise the traditional preservation methods.