Emergence of genetic variants with increased resistance/tolerance to natural antimicrobials, such as essential oils, has been previously evidenced; however, it is unknown whether mutagenesis follows a general or a specific pattern. For this purpose, we carried out four adaptive laboratory evolutions (ALE) in parallel of Salmonella enterica Typhimurium with carvacrol. After 10 evolution steps, we selected and characterized one colony from each lineage (SeCarA, SeCarB, SeCarC, and SeCarD). Phenotypic characterization of the four evolved strains revealed enhanced survival to lethal treatments; two of them (SeCarA and SeCarB) showed an increase of minimum inhibitory concentration of carvacrol and a better growth fitness in the presence of carvacrol compared to wild-type strain. Whole genome sequencing revealed 10 mutations, of which four (rrsH, sseG, wbaV, and flhA) were present in more than one strain, whereas six (nirC, fliH, lon, rob, upstream yfhP, and upstream argR) were unique to individual strains. Single-mutation genetic constructs in SeWT confirmed lon and rob as responsible for the increased resistance to carvacrol as well as to antibiotics (ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, rifampicin, tetracycline, and trimethoprim). wbaV played an important role in increased tolerance against carvacrol and chloramphenicol, and flhA in cross-tolerance to heat treatments. As a conclusion, no common phenotypical or genotypical pattern was observed in the isolated resistant variants of Salmonella Typhimurium emerged under carvacrol stress. Furthermore, the demonstration of cross-resistance against heat and antibiotics exhibited by resistant variants raises concerns regarding food safety. Key points: • Stable resistant variants of Salmonella Typhimurium emerged under carvacrol stress • No common pattern of mutagenesis after cyclic exposures to carvacrol was observed • Resistant variants to carvacrol showed cross-resistance to heat and to antibiotics Graphical

Essential oils and their constituents, such as carvacrol, are potential food preservatives because of their great antimicrobial properties. However, the long-term effects of these compounds are unknown and raise the question of whether resistance to these antimicrobials could emerge. This work aims to evaluate the occurrence of genetic resistant variants (RVs) in Listeria monocytogenes EGD-e by exposure to carvacrol. Two protocols were performed for the RVs selection: (a) by continuous exposure to sublethal doses, where LmSCar was isolated, and (b) by reiterative exposure to short lethal treatments of carvacrol, where LmLCar was isolated. Both RVs showed an increase in carvacrol resistance. Moreover, LmLCar revealed an increased cross-resistance to heat treatments at acid conditions and to ampicillin. Whole-genome sequencing identified two single nucleotide variations in LmSCar and three non-silent mutations in LmLCar. Among them, those located in the genes encoding the transcriptional regulators RsbT (in LmSCar) and ManR (in LmLCar) could contribute to their increased carvacrol resistance. These results provide information regarding the mode of action of this antimicrobial and support the importance of knowing how RVs appear. Further studies are required to determine the emergence of RVs in food matrices and their impact on food safety.

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.