Abstract
Due to their excellent antimicrobial properties, essential oils (EO) have been proposed as potential preservatives for certain kinds of foods, such as dairy products. However, the occurrence of pathogenic populations that are resistant to EOs could pose a health risk. This report seeks to assess the emergence of resistant populations in Listeria monocytogenes EGD-e growth at 37 °C under selective pressure of Thymbra capitata EO (TCO), to characterise their resistance in laboratory media, and to identify their genotypic changes, as well as to evaluate the resistance in skimmed milk. TCO cyclic treatment allowed the isolation of two L. monocytogenes EGD-e resistant strains against the EO: LmSTCO by sublethal doses (75 µL/L TCO) and LmLTCO by lethal doses (300 µL/L TCO) after 20 and 30 cycles, respectively. Both strains displayed an increase of the minimum inhibitory and bactericidal concentration against TCO and a higher survival rate after lethal treatments than the wild-type strain (LmWT). Growth kinetics revealed a better adaptation of LmSTCO in presence of TCO, while LmLTCO grew more slowly compared to LmWT, even in the absence of the antimicrobial. Moreover, a slight increase in cross-resistance to antibiotics was observed: LmSTCO to β-lactams and LmLTCO to a series of broad-spectrum antibiotics. The genomic study revealed one sole nucleotide change in LmSTCO located in plsC gene codifying an enzyme involved in the production of phosphatidic acid, a precursor in cell membrane synthesis. Five genetic variations were found in LmLTCO: among them, the deletion of an ATP-synthesis system involved in slowing bacterial growth. Inhibition and inactivation assays in skimmed milk confirmed the increased resistance of both strains, thereby indicating a safety risk in case these strains emerge in the food chain. These results strongly suggest that the occurrence of such resistances should be taken into account in order to ensure the efficacy of natural antimicrobials in the design of food preservation strategies.
Highlights
Natural antimicrobials are emerging as an alternative to chemically synthesized food preservatives, which are subject to greater restrictions and are increasingly rejected by consumers (Carocho, Barreiro, Morales, & Ferreira, 2014)
After carrying out the Thymbra capitata Essential oils (EOs) (TCO) evolution assays with L. monocytogenes EGD-e (LmWT) with two different protocols: cyclic exposure to a)
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) results of the 5 colonies selected from the evolution assays were grouped in the same cell because they showed the same values for both parameters
Summary
Natural antimicrobials are emerging as an alternative to chemically synthesized food preservatives, which are subject to greater restrictions and are increasingly rejected by consumers (Carocho, Barreiro, Morales, & Ferreira, 2014). Essential oils (EOs) and their individual constituents (ICs) have demonstrated excellent antimicrobial and antioxidant prop erties (Faleiro & Miguel, 2020) that point toward their potential use in the food industry (Quinto et al, 2019). Their current use in food pres ervation is limited by some drawbacks. Prolonged evolution assays at sublethal and lethal doses demonstrated the emergence of resistant and tolerant strains against ICs, such as carvacrol, citral and limonene oxide, in food pathogens: Escherichia coli (Chueca et al, 2016), Salmonella enterica (Berdejo, Me rino, Pagan, García-Gonzalo, & Pagan, 2020) and Staphylococcus aureus (Berdejo et al, 2019). It should be noted that “resistance” is the ability of bacteria to replicate and not just survive in the presence of a drug, whereas “tolerance” is the general ability of a population to survive longer treatments (Balaban et al, 2019)
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