(+)-limonene is a lipophilic antimicrobial compound, extracted from citrus fruits' essential oils, that is used as a flavouring agent and organic solvent by the food industry. A recent study has proposed a common and controversial mechanism of cell death for bactericidal antibiotics, in which hydroxyl radicals ultimately inactivated cells. Our objective was to determine whether the mechanism of Escherichia coli MG1655 inactivation by (+)-limonene follows that of bactericidal antibiotics. A treatment with 2,000 μL/L (+)-limonene inactivated 4 log10 cycles of exponentially growing E. coli cells in 3 hours. On one hand, an increase of cell survival in the ΔacnB mutant (deficient in a TCA cycle enzyme), or in the presence of 2,2′-dipyridyl (inhibitor of Fenton reaction by iron chelation), thiourea, or cysteamine (hydroxyl radical scavengers) was observed. Moreover, the ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) was more sensitive to (+)-limonene. Thus, this indirect evidence indicates that the mechanism of exponentially growing E. coli cells inactivation by 2,000 μL/L (+)-limonene is due to the TCA cycle and Fenton-mediated hydroxyl radical formation that caused oxidative DNA damage, as observed for bactericidal drugs. However, several differences have been observed between the proposed mechanism for bactericidal drugs and for (+)-limonene. In this regard, our results demonstrated that E. coli inactivation was influenced by its physiological state and the drug's concentration: experiments with stationary-phase cells or 4,000 μL/L (+)-limonene uncovered a different mechanism of cell death, likely unrelated to hydroxyl radicals. Our research has also shown that drug's concentration is an important factor influencing the mechanism of bacterial inactivation by antibiotics, such as kanamycin. These results might help in improving and spreading the use of (+)-limonene as an antimicrobial compound, and in clarifying the controversy about the mechanism of inactivation by bactericidal antibiotics.

The improvement of food preservation by using essential oils (EOs) and their individual constituents (ICs) is attracting enormous interest worldwide. Until now, researchers considered that treatments with such antimicrobial compounds did not induce bacterial resistance via a phenotypic (i.e., transient) response. Nevertheless, the emergence of genotypic (i.e., stable) resistance after treatment with these compounds had not been previously tested. Our results confirm that growth of Escherichia coli MG1655 in presence of sub-inhibitory concentrations of the ICs carvacrol, citral, and (+)-limonene oxide do not increase resistance to further treatments with either the same IC (direct resistance) or with other preservation treatments (cross-resistance) such as heat or pulsed electric fields (PEF). Bacterial mutation frequency was likewise lower when those IC's were applied; however, after 10 days of re-culturing cells in presence of sub-inhibitory concentrations of the ICs, we were able to isolate several derivative strains (i.e., mutants) displaying an increased minimum inhibitory concentration to those ICs. Furthermore, when compared to the wild type (WT) strain, they also displayed direct resistance and cross-resistance. Derivative strains selected with carvacrol and citral also displayed morphological changes involving filamentation along with cell counts at late-stationary growth phase that were lower than the WT strain. In addition, co-cultures of each derivative strain with the WT strain resulted in a predominance of the original strain in absence of ICs, indicating that mutants would not out-compete WT cells under optimal growth conditions. Nevertheless, growth in the presence of ICs facilitated the selection of these resistant mutants. Thus, as a result, subsequent food preservation treatments of these bacterial cultures might be less effective than expected for WT cultures. In conclusion, this study recommends that treatment with ICs at sub-inhibitory concentrations should be generally avoided, since it could favor the emergence of hyper-resistant strains. To ascertain the true value of EOs and their ICs in the field of food preservation, further research thus needs to be conducted on the induction of increased transient and stable bacterial resistance via such antimicrobial compounds, as revealed in this study.

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Increased bacterial resistance to food preservation technologies represents a risk for food safety and shelf-life. The use of natural antimicrobials, such as essential oils (EOs) and their individual constituents (ICs), has been proposed to avoid the generation of antimicrobial resistance. However, prolonged application of ICs might conceivably lead to the emergence of resistant strains. Hence, this study was aimed toward applying sub-inhibitory doses of the ICs carvacrol, citral, and (+)-limonene oxide to Staphylococcus aureus USA300, in order to evaluate the emergence of resistant strains and to identify the genetic modifications responsible for their increased resistance. Three stable-resistant strains, CAR (from cultures with carvacrol), CIT (from cultures with citral), and OXLIM (from cultures with (+)-limonene oxide) were isolated, showing an increased resistance against the ICs and a higher tolerance to lethal treatments by ICs or heat. Whole-genome sequencing revealed in CAR a large deletion in a region that contained genes encoding transcriptional regulators and metabolic enzymes. CIT showed a single missense mutation in aroC (N187K), which encodes for chorismate synthase; and in OXLIM a missense mutation was detected in rpoB (A862V), which encodes for RNA polymerase subunit beta. This study provides a first detailed insight into the mechanisms of action and S. aureus resistance arising from exposure to carvacrol, citral, and (+)-limonene oxide.