Carvacrol Selective Pressure Allows the Occurrence of Genetic Resistant Variants of Listeria monocytogenes EGD-e

Abstract

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.

Keywords: Listeria monocytogenes; antibiotic resistance; carvacrol; evolution assays; heat treatments; resistant variants; whole-genome sequencing.

Figure 1

Survival curves of Listeria monocytogenes EGD-e (●; LmWT) and its evolved strains, LmSCar (■; exposed to prolonged sublethal doses of carvacrol) and LmLCar (▲; cyclically exposed to short lethal treatments of carvacrol), to 200-µL/L carvacrol treatment at pH 4.0 (A) and 300-µL/L carvacrol treatment at pH 7.0 (B). Data are means ± standard deviations (error bars) obtained from at least three independent experiments. The dashed line represents the detection limit (−5.5 log₁₀ N_t/N₀).

Figure 2

Survival curves of Listeria monocytogenes EGD-e (●; LmWT) and its evolved strains, LmSCar (■; exposed to prolonged sublethal doses of carvacrol) and LmLCar (▲; cyclically exposed to short lethal treatments of carvacrol), to heat treatments: 54 °C at pH 4.0 (A); and 58 °C at pH 7.0 (B). Data are means ± standard deviations (error bars) obtained from at least three independent experiments. The dashed line represents the detection limit (−5.5 log₁₀ N_t/N₀).