Sub-Inhibitory Doses of Individual Constituents of Essential Oils Can Select for Staphylococcus aureus Resistant Mutants.

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.

Whole-Genome Sequencing and Genetic Analysis Reveal Novel Stress Responses to Individual Constituents of Essential Oils in Escherichia coli.

Food preservation by the use of essential oils (EOs) is being extensively studied because of the antimicrobial properties of their individual constituents (ICs). Three resistant mutants (termed CAR, CIT, and LIM) of Escherichia coli MG1655 were selected by subculturing with the ICs carvacrol, citral, and (+)-limonene oxide, respectively. These derivative strains showed increased MIC values of ICs and concomitantly enhanced resistance to various antibiotics (ampicillin, trimethoprim, chloramphenicol, tetracycline, kanamycin, novobiocin, norfloxacin, cephalexin, and nalidixic acid) compared to those for the parental strain (wild type [WT]). Whole-genome sequencing (WGS) of these hyperresistant strains permitted the identification of single nucleotide polymorphisms (SNPs) and deletions in comparison to the WT. In order to analyze the contribution of these mutations to the increased antimicrobial resistance detected in hyperresistant strains, derivative strains were constructed by allelic reversion. A role of the SoxR D137Y missense mutation in CAR was confirmed by growth in the presence of some ICs and antibiotics and by its tolerance to ICs but not to lethal heat treatments. In CIT, increased resistance relied on contributions by several detected SNPs, resulting in a frameshift in MarR and an in-frame GyrB ΔG157 mutation. Finally, both the insertion resulting in an AcrR frameshift and large chromosomal deletions found in LIM were correlated with the hyperresistant phenotype of this strain. The nature of the obtained mutants suggests intriguing links to cellular defense mechanisms previously implicated in antibiotic resistance.IMPORTANCE The antimicrobial efficacy of ICs has been proven over the years, together with their potential to improve traditional heat treatments by reducing treatment intensity and, consequently, adverse effects on food quality. However, the mechanisms of bacterial inactivation by ICs are still not well understood, in contrast to antibiotics. We performed WGS of three E. coli strains that are hyperresistant to ICs. The information provided detailed insight into the mechanisms of bacterial resistance arising from exposure to carvacrol, citral, and (+)-limonene oxide. Future experiments will undoubtedly yield additional insights into genes and pathways contributing to the acquisition of endogenous resistance to ICs.

Global transcriptional response of Escherichia coli MG1655 cells exposed to the oxygenated monoterpenes citral and carvacrol.

DNA microarrays were used to study the mechanism of bacterial inactivation by carvacrol and citral. After 10-min treatments of Escherichia coli MG1655 cells with 100 and 50ppm of carvacrol and citral, 76 and 156 genes demonstrated significant transcriptional differences (p≤0.05), respectively. Among the up-regulated genes after carvacrol treatment, we found gene coding for multidrug efflux pumps (acrA, mdtM), genes related to phage shock response (pspA, pspB, pspC, pspD, pspF and pspG), biosynthesis of arginine (argC, argG, artJ), and purine nucleotides (purC, purM). In citral-treated cells, transcription of purH and pyrB and pyrI was 2 times higher. Deletion of several differentially expressed genes confirmed the role of ygaV, yjbO, pspC, sdhA, yejG and ygaV in the mechanisms of E. coli inactivation by carvacrol and citral. These results would indicate that citral and carvacrol treatments cause membrane damage and activate metabolism through the production of nucleotides required for DNA and RNA synthesis and metabolic processes. Comparative transcriptomics of the response of E. coli to a heat treatment, which caused a significant change of the transcription of 1422 genes, revealed a much weaker response to both individual constituents of essential oils (ICs).·Thus, inactivation by citral or carvacrol was not multitarget in nature.

Emergence of Hyper-Resistant Escherichia coli MG1655 Derivative Strains after Applying Sub-Inhibitory Doses of Individual Constituents of Essential Oils.

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.

Inactivation of spoiling microorganisms in apple juice by a combination of essential oils' constituents and physical treatments.

A combination of different hurdles, such as mild heat (54 ℃ for 10 min) or pulsed electric field (25 pulses; 25 kV/cm; 3.35 kJ/cm per pulse) treatments and essential oils constituents (carvacrol, citral, and (+)-limonene), to reduce spoiling bacteria and yeasts in apple juice was evaluated. For this purpose, the heat and pulsed electric field resistances of five strains of Leuconostoc spp. and five Saccharomyces spp. strains were assayed, achieving different inactivation levels for each treatment and strain. For instance, Leuconostoc fallax 74, the most heat-resistant strain, was the second-most sensitive strain to pulsed electric field. The most resistant strains were exposed to combined processes of heat or pulsed electric field and 0.2 µl/ml essential oils constituents. The combination of heat and essential oils constituents proved to be synergistic against both microorganisms in apple juice. The most effective was the combination of mild heat and carvacrol, which caused the inactivation of 99% of L. fallax 74 and 99.99% of Saccharomyces cerevisiae CECT 1172 cells. Therefore, this study shows the great potential of carvacrol, citral, and (+)-limonene in combined treatments with mild heat to achieve a higher degree of inactivation of spoiling microorganisms in apple juice, and thus, to extend its shelf life.

Influence of general stress-response alternative sigma factors σ(S) (RpoS) and σ(B) (SigB) on bacterial tolerance to the essential oils from Origanum vulgare L. and Rosmarinus officinalis L. and pulsed electric fields.

This study assessed the influence of general stress-response alternative sigma factors RpoS (σ(S)) and SigB (σ(B)) on tolerance of Escherichia coli (E. coli MG1655 and its isogenic mutant E. coli MG1655 ΔrpoS) and Listeria monocytogenes (L. monocytogenes EGD-e and its isogenic mutant L. monocytogenes EGD-e ΔsigB) to the essential oils (EOs) from Origanum vulgare L.-oregano (OVEO) and Rosmarinus officinalis L.-rosemary (ROEO), as well as the changes in tolerance of parental and ΔrpoS and ΔsigB mutant strains to OVEO, ROEO and pulsed electric fields (PEF) following overnight exposure to subinhibitory concentrations (1/2×minimum inhibitory concentration-MIC) of each tested EO. MIC values of OVEO and ROEO against the mutant cells were usually lower than those found against the parental cells. Survivor curves showed that mutant cells were more sensitive to these EOs than parental cells. The recovery of survivors in selective media showed a greater proportion of cells sublethally injured at their cell envelopes in the mutant strains compared with the parental strains. Induction of increased direct-tolerance to OVEO and ROEO or cross-tolerance to PEF was not observed after pre-exposure of parental and mutant cells to EOs. Otherwise, parental and mutant cells of E. coli and L. monocytogenes pre-exposed to OVEO or ROEO showed decreased tolerance when further treated with the homologous stressing agent at 2×MIC. Still, mutant cells pre-exposed to OVEO or ROEO showed lower tolerance to PEF than parental strains. These results showed the influence of σ(S) and σ(B) in tolerance of single strains of E. coli and L. monocytogenes, respectively, to OVEO and ROEO. Moreover, the deletion of σ(S) and σ(B) resulted in decreased tolerance to OVEO, ROEO or PEF in tested strains following exposure to OVEO or ROEO at a subinhibitory concentration.

Oxygenated monoterpenes citral and carvacrol cause oxidative damage in Escherichia coli without the involvement of tricarboxylic acid cycle and Fenton reaction.

Oxygenated monoterpenes citral and carvacrol are common constituents of many essential oils (EOs) that have been extensively studied as antimicrobial agents but whose mechanisms of microbial inactivation have not been totally elucidated. A recent study described a mechanism of Escherichia coli death for (+)-limonene, a hydrocarbon monoterpene also frequently present in EOs, similar to the common mechanism proposed for bactericidal antibiotics. This mechanism involves the formation of Fenton-mediated hydroxyl radical, a reactive oxygen species (ROS), via tricarboxylic acid (TCA) cycle, which would ultimately inactivate cells. Our objective was to determine whether E. coli MG1655 inactivation by citral and carvacrol follows a similar mechanism of cell death. Challenging experiments with 300µL/L citral and 100µL/L carvacrol inactivated at least 2.5log10cycles of exponentially growing cells in 3h under aerobic conditions. The presence of thiourea (an ROS scavenger) reduced cell inactivation in 2log10cycles, demonstrating the role of ROS in cell death. Decreased resistance of a ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) indicated that citral and carvacrol caused oxidative damage to DNA. Although the mechanism of E. coli inactivation by carvacrol and citral was similarly mediated by ROS, their formation did not follow the same pathways described for (+)-limonene and bactericidal drugs because neither Fenton reaction nor NADH production via the TCA cycle was involved in cell death. Moreover, further experiments demonstrated antimicrobial activity of citral and carvacrol in anaerobic environments without the involvement of ROS. As a consequence, cell death by carvacrol and citral in anaerobiosis follows a different mechanism than that observed under aerobic conditions. These results demonstrated a different mechanism of inactivation by citral and carvacrol with regard to (+)-limonene and bactericidal antibiotics, indicating the complexity of the mechanisms of bacterial inactivation among EO constituents. Advancements in the description of these mechanisms will help in extending and improving the use of these compounds as natural antimicrobials.

Differential mechanism of Escherichia coli Inactivation by (+)-limonene as a function of cell physiological state and drug's concentration.

(+)-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.