Evolutionary trade-off between heat shock resistance, growth at high temperature, and virulence expression in Salmonella Typhimurium.

Understanding the evolutionary dynamics of foodborne pathogens throughout our food production chain is of utmost importance. In this study, we reveal that Salmonella Typhimurium can readily and reproducibly acquire vastly increased heat shock resistance upon repeated exposure to heat shock. Counterintuitively, this boost in heat shock resistance was invariantly acquired through loss-of-function mutations in the dnaJ gene, encoding a heat shock protein that acts as a molecular co-chaperone of DnaK and enables its role in protein folding and disaggregation. As a trade-off, however, the acquisition of heat shock resistance inevitably led to attenuated growth at 37°C and higher temperatures. Interestingly, loss of DnaJ also downregulated the activity of the master virulence regulator HilD, thereby lowering the fraction of virulence-expressing cells within the population and attenuating virulence in mice. By connecting heat shock resistance evolution to attenuation of HilD activity, our results confirm the complex interplay between stress resistance and virulence in Salmonella Typhimurium. IMPORTANCE: Bacterial pathogens such as Salmonella Typhimurium are equipped with both stress response and virulence features in order to navigate across a variety of complex inhospitable environments that range from food-processing plants up to the gastrointestinal tract of its animal host. In this context, however, it remains obscure whether and how adaptation to one environment would obstruct fitness in another. In this study, we reveal that severe heat stress counterintuitively, but invariantly, led to the selection of S. Typhimurium mutants that are compromised in the activity of the DnaJ heat shock protein. While these mutants obtained massively increased heat resistance, their virulence became greatly attenuated. Our observations, therefore, reveal a delicate balance between optimal tuning of stress response and virulence features in bacterial pathogens.

Adaptive evolution of Salmonella Typhimurium LT2 exposed to carvacrol lacks a uniform pattern.

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.

Minimal Processing Technologies for Production and Preservation of Tailor-Made Foods§.

Tailor-made foods, also known as foods with programmable properties, are specialised systems with unique composition prepared by different methods, using the known mechanisms of action of their bioactive ingredients. The development of tailor-made foods involves the evaluation of individual components, including bioactive substances derived from waste products of other productions, such as essential oils. These components are evaluated both individually and in combination within food compositions to achieve specific functionalities. This review focuses on the application of minimal processing technologies for the production and preservation of tailor-made foods. It examines a range of approaches, including traditional and emerging technologies, as well as novel ingredients such as biomolecules from various sources and microorganisms. These approaches are combined according to the principles of hurdle technology to achieve effective synergistic effects that enhance food safety and extend the shelf life of tailor-made foods, while maintaining their functional properties.

Phenotypic and Genotypic Comparison of Antimicrobial-Resistant Variants of Escherichia coli and Salmonella Typhimurium Isolated from Evolution Assays with Antibiotics or Commercial Products Based on Essential Oils.

On account of the widespread development and propagation of antimicrobial-resistant (AMR) bacteria, essential oils (EOs) have emerged as potential alternatives to antibiotics. However, as already observed for antibiotics, recent studies have raised concerns regarding the potential emergence of resistant variants (RVs) to EOs. In this study, we assessed the emergence of RVs in Escherichia coli and Salmonella enterica Typhimurium after evolution assays under extended exposure to subinhibitory doses of two commercial EOs (AEN and COLIFIT) as well as to two antibiotics (amoxicillin and colistin). Phenotypic characterization of RVs from evolution assays with commercial EOs yielded no relevant increases in the minimum inhibitory concentration (MIC) of E. coli and did not even modify MIC values in S. Typhimurium. Conversely, RVs of E. coli and S. Typhimurium isolated from evolution assays with antibiotics showed increased resistance. Genotypic analysis demonstrated that resistance to commercial EOs was associated with enhanced protection against oxidative stress and redirection of cell energy toward efflux activity, while resistance to antibiotics was primarily linked to modifications in the cell binding sites of antibiotics. These findings suggest that AEN and COLIFIT could serve as safe alternatives to antibiotics in combating the emergence and dissemination of antimicrobial resistance within the agrifood system.

Oil-in-Water Pickering Emulsions Stabilized with Nanostructured Biopolymers: A Venue for Templating Bacterial Cellulose.

Pickering emulsions (PEs) differ from conventional emulsions in the use of solid colloidal particles as stabilizing agents instead of traditional amphiphilic molecules. Nanostructured biopolymers (NBs) emerge as a promising alternative for PE stabilization owing to their remarkable biocompatibility, abundant availability, and low cost. To explore this potential, a study is herein presented, in which cellulose nanocrystals (CNCs), both type I and type II allomorphs, and chitin nanocrystals (ChNCs) were used for stabilizing oil-in-water PEs prepared by the use of ultrasound. Sunflower oil was selected as the oil phase as it offers the advantages of being edible, renewable, and inexpensive. By utilizing ζ-potential, static light diffraction, and visual observations, we determined the optimal oil/water ratio for each type of NB to obtain stable emulsions after 14 days. The optimized PEs were used to form bacterial nanocellulose composites through emulsion templating. To our knowledge, this study represents a pioneering work in exploiting oil-in-water PEs for this approach. Additionally, it entails the first utilization of nonmercerized type II CNCs as stabilizers for PEs, while also establishing a direct comparison among the most relevant NBs. The resulting composites exhibited a unique morphology, composed of larger pores compared to standard bacterial nanocellulose aerogels. These findings highlight the notable potential of NBs as stabilizers for PEs and their ability to generate green nanocomposites with tailored properties.

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

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.

Salmonella enterica serovar Typhimurium genetic variants isolated after lethal treatment with Thymbra capitata essential oil (TCO) showed increased resistance to TCO in milk.

The high prevalence of Salmonella enterica in milk poses a risk of considerable concern in the preservation of certain dairy products, mainly those elaborated from raw milk. Essential oils (EOs) have been proposed as a promising food preservative for such products due to their strong antimicrobial properties. Additionally, these natural antimicrobials have been shown to be effective against multi-drug resistant strains. They can thus also be utilized to prevent the dissemination of antimicrobial resistances (AMR). However, recent evidence of the development of bacterial resistance under EO treatments may call their use into question. This study sought to assess the emergence of antimicrobial resistant genetic variants of S. enterica serovar Typhimurium from survivors after cyclic exposure to lethal doses (>5 log10 cycles of inactivation) of Thymbra capitata EO (TCO), in order to evaluate the impact that it could have on milk preservation, to ascertain whether cross-resistance to antibiotics occurs, and to identify the genomic changes responsible for their phenotype. Isolated strains by TCO (SeTCO) showed a two-fold increase in minimum inhibitory and bactericide concentrations (MIC and MBC) of TCO compared to Salmonella enterica serovar Typhimurium wild-type strain (SeWT) in laboratory growth medium, as well as a greater adaptation and growth rate in the presence of the EOs and a higher survival to TCO treatments in buffers of pH 4.0 and 7.0. The increased resistance of SeTCO was confirmed in skimmed milk: 300 µL/L TCO reduced only 1 log10 cycle of SeTCO population, whereas it inactivated more than 5 log10 cycles in SeWT. Moreover, SeTCO showed an increased cross-resistance against aminoglycosides, quinolones and tetracyclines. Whole genome sequencing revealed 5 mutations in SeTCO: 2 in genes involved in O-antigens synthesis (rfbV and rfbX), 2 in genes related to adaptation to the growing medium (trkA and glpK), and 1 in a redox-sensitive transcriptional regulator (soxR). The phenotypic characterization of a constructed SeWT strain with mutant soxRSeTCO demonstrated that the mutation of soxR was the main cause of the increased resistance and tolerance observed in SeTCO against TCO and antibiotics. The emergence of resistant strains against EOs might jeopardize their use as food preservatives. Further studies will thus be required to determine under which conditions such resistant strains might occur, and to assess the food risk they may pose, as well as to ascertain their impact on the spread of AMR.

Emerging mutant populations of Listeria monocytogenes EGD-e under selective pressure of Thymbra capitata essential oil question its use in food preservation.

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 ß-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.

Modified cyclodextrin type and dehydration methods exert a significant effect on the antimicrobial activity of encapsulated carvacrol and thymol.

BACKGROUND: The antimicrobial activity of essential oils and their constituents has led to increasing interest in using them as natural preservative agents. However, their high sensitivity to light and oxygen, their volatility and their low aqueous solubility are all obstacles to their application in the food, cosmetic or pharmaceutical industries. Encapsulation in cyclodextrins (CDs) is a solution for the application of such essential oils. RESULTS: The complexation of carvacrol and thymol with hydroxypropyl (HP)-α-, HP-ß- and HP-γ-CD, the behavior of the solid complexes prepared by freeze-drying and spray-drying methods and the antibacterial activity of solid complexes were studied. Kc values of HP-α- and HP-γ-CD complexes with carvacrol (118.4 and 365.7 L mol-1 ) and thymol (112.5 and 239.7 L mol -1 ) were far lower than those observed for HP-ß-CD complexes with carvacrol (2268.2 L mol -1 ) and thymol (881.6 L mol -1 ). The lower stability of HP-α- and HP-γ-CD complexes increased the release of compounds, thereby affecting the antimicrobial activity of carvacrol and thymol to a lesser extent than complexation with HP-ß-CD, normally used in the encapsulation of carvacrol and thymol. HP-ß-CD encapsulation of carvacrol and thymol markedly reduced their antimicrobial activity. The freeze-drying method barely affected the antimicrobial activity of carvacrol and thymol after encapsulation, while spray drying could be considered for the production of solid complexes in combination with the appropriate CD. CONCLUSIONS: It was thus demonstrated that HP-α- and HP-γ-CD are very suitable alternatives for the encapsulation of carvacrol and thymol with the purpose of preserving their bacteriostatic and bactericidal activities. © 2020 Society of Chemical Industry.

Incubation with a Complex Orange Essential Oil Leads to Evolved Mutants with Increased Resistance and Tolerance.

Emergence of strains with increased resistance/tolerance to natural antimicrobials was evidenced after cyclic exposure to carvacrol, citral, and (+)-limonene oxide. However, no previous studies have reported the development of resistance and tolerance to complex essential oils (EOs). This study seeks to evaluate the occurrence of Staphylococcus aureus strains resistant and tolerant to a complex orange essential oil (OEO) after prolonged cyclic treatments at low concentrations. Phenotypic characterization of evolved strains revealed an increase of minimum inhibitory and bactericidal concentration for OEO, a better growth fitness in presence of OEO, and an enhanced survival to lethal treatments, compared to wild-type strain. However, no significant differences (p > 0.05) in cross-resistance to antibiotics were observed. Mutations in hepT and accA in evolved strains highlight the important role of oxidative stress in the cell response to OEO, as well as the relevance of the cell membrane in the cell response to these natural antimicrobials. This study demonstrates the emergence of S. aureus strains that are resistant and tolerant to EO (Citrus sinensis). This phenomenon should be taken into account to assure the efficacy of natural antimicrobials in the design of food preservation strategies, in cleaning and disinfection protocols, and in clinical applications against resistant bacteria.

Chitosan nanoemulsions of cold-pressed orange essential oil to preserve fruit juices.

Sweet orange essential oil is obtained from the peels of Citrus sinensis (CSEO) by cold pressing, and used as a valuable product by the food industry. Nanoencapsulation is known as a valid strategy to improve chemical stability, organoleptic properties, and delivery of EO-based products. In the present study we encapsulated CSEO using chitosan nanoemulsions (cn) as nanocarrier, and evaluated its antimicrobial activity in combination with mild heat, as well as its sensorial acceptability in orange and apple juices. CSEO composition was analyzed by GC-MS, and 19 components were identified, with limonene as the predominant constituent (95.1%). cn-CSEO was prepared under low shear conditions and characterized according to droplet size (<60 nm) and polydispersity index (<0.260 nm). Nanoemulsions were stable for at least 3 months at 4 ± 2 °C. cn-CSEO were compared with suspensions of CSEO (s-CSEO) (0.2 µL of CSEO/mL) in terms of antibacterial activity in combination with mild heat (52 °C) against Escherichia coli O157:H7 Sakai. cn-CSEO displayed a greater bactericidal activity than s-CSEO at pH 7.0 and pH 4.0. The validation in fruit juices showed an improved bactericidal effect of cn-CSEO in comparison with s-CSEO when combined with mild heat in apple juice, but not in orange juice. In both juices, the combination of CSEO and mild heat exerted synergistic lethal effects, reducing the treatment time to cause the inactivation of up to 5 Log10 cycles of E. coli O157:H7 Sakai cells. Finally, the sensory characteristics of both juices were acceptable either when using s-CSEO or CSEO nanoemulsified with chitosan. Therefore, as a promising carrier for lipophilic substances, the encapsulation of EOs with chitosan nanoemulsions might represent an advantageous alternative when combined with mild heat to preserve fruit juices.

Genetic Variants and Phenotypic Characteristics of Salmonella Typhimurium-Resistant Mutants after Exposure to Carvacrol.

The emergence of antimicrobial resistance has raised questions about the safety of essential oils and their individual constituents as food preservatives and as disinfection agents. Further research is required to understand how and under what conditions stable genotypic resistance might occur in food pathogens. Evolution experiments on Salmonella Typhimurium cyclically exposed to sublethal and lethal doses of carvacrol permitted the isolation of SeSCar and SeLCar strains, respectively. Both evolved strains showed a significant increase in carvacrol resistance, assessed by minimum inhibitory and bactericidal concentrations, the study of growth kinetics in the presence of carvacrol, and the evaluation of survival under lethal conditions. Moreover, antibiotic susceptibility tests revealed a development of SeLCar resistance to a wide range of antibiotics. Whole genome sequencing allowed the identification of single nucleotide variations in transcriptional regulators of oxidative stress-response: yfhP in SeSCar and soxR in SeLCar, which could be responsible for the increased resistance by improving the response to carvacrol and preventing its accumulation inside the cell. This study demonstrates the emergence of S. Typhimurium-resistant mutants against carvacrol, which might pose a risk to food safety and should therefore be considered in the design of food preservation strategies, or of cleaning and disinfection treatments.

Combination of mild heat and plant essential oil constituents to inactivate resistant variants of Escherichia coli in buffer and in coconut water.

The growing demand for minimally processed foods with clean labels has stimulated research into mild processing methods and natural antimicrobials to replace intensive heating and conventional preservatives, respectively. However, we have previously demonstrated that repetitive exposure of some bacteria to mild heat or subinhibitory concentrations of essential oil constituents (EOCs) may induce the emergence of mutants with increased resistance to these treatments. Since the combination of mild heat with some EOCs has a synergistic effect on microbial inactivation, we evaluated the potential of such combinations against our resistant E. coli mutants. While citral, carvacrol and t-cinnamaldehyde synergistically increased heat inactivation (53.0 °C, 10 min) of the wild-type MG1655 suspended in buffer, only the combination with carvacrol (200 µl/l) was able to mitigate the increased resistance of all the mutants. Moreover, the combination of heat and carvacrol acted synergistically inactivating heat-resistant variants of E. coli O157:H7 (ATCC 43888). This combined treatment could synergistically achieve more than 5 log10 reductions of the most resistant mutants in coconut water, although the temperature had to be raised to 57.0 °C. Therefore, the combination of mild heat with carvacrol appears to hold promise for mild processing, and it is expected to counteract the development of heat resistance.

Control of Autochthonous Spoilage Lactic Acid Bacteria in Apple and Orange Juices by Sensorially Accepted Doses of Citrus Spp. Essential Oils Combined with Mild Heat Treatments.

This study assessed the compromised acceptance threshold (CAT) and rejection threshold (RT) of Citrus lemon (CLEO) and Citrus reticulata essential oil (CREO) in apple and orange juices. The efficacy of CLEO and CREO concentrations below the RT were evaluated alone and combined with mild heat treatment (MHT) (54 °C, up to 12 min) to inactivate the autochthonous spoilage bacteria Lactobacillus brevis, Lactobacillus plantarum, and Leuconostoc mesenteroides in apple and orange juices. The CAT of CLEO and CREO varied from 0.15 to 0.17 µL/mL in orange and apple juices. The RT of CLEO was approximately 0.58 µL/mL in apple and orange juices, and the RT of CREO was 0.68 µL/mL in both juices. When CLEO and CREO were assayed alone, the highest concentration (0.50 µL/mL) decreased counts of all strains approximately 2 log10 CFU/mL after 12 min of exposure to 54 °C. All concentrations of CLEO or CREO in combination with MHT acted synergistically against L. brevis, L. plantarum, and L. mesenteroides. Decreases in counts varied with the strain, CLEO and CREO concentrations, juice type, and exposure time to the combined treatment. CREO was more effective than CLEO in combination with MHT against the strains in apple and orange juices. Effective combinations of CLEO or CREO with MHT to control the autochthonous spoilage bacteria did not compromise the quality parameters (°Brix, pH, and titratable acidity) that characterize unsweetened juices. These results indicate CLEO or CREO at concentrations below the sensory RT in combination with MHT as a feasible technology to control autochthonous spoilage bacteria in fresh fruit juices. PRACTICAL APPLICATION: The present study provides novel information concerning the efficacy of sensorially accepted doses of CLEO and CREO combined with MHT against autochthonous spoilers in fruit juice. The valuable synergistic effects that can be observed when combining CLEO and CREO with MHT reveal a feasible preservation technology and alternative to traditional treatments that are successful because they help reduce treatment intensity, thereby avoiding adverse effects on the sensory, physicochemical, and nutritional properties of these products.

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.

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.