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.

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.

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.

Growth fitness, virulence, and heat tolerance of Salmonella Typhimurium variants resistant to food preservation methods.

To study potential ramifications of antimicrobial resistance, we carried out adaptive laboratory evolution assays (ALE) to isolate three resistant variants (RVs) of Salmonella enterica Typhimurium, employing three different types of food preservation methods: 1) an emergent technology, plasma-activated water (PAW), leading to variant RV-PAW; a traditional method, heat, leading to variant RV-HT, and a natural antimicrobial compound, carvacrol, leading to variant RV-CAR. The variant resistant to plasma-activated water, RV-PAW, had mutations in rpoA and rpoD; it showed increased tolerance to heat in orange juice but ultimately did not pose a significant threat, as it exhibited a fitness cost at refrigeration temperature (8 °C), whereas its virulence against Caenorhabditis elegans decreased. The variant resistant to heat, RV-HT, had mutations in flhC, dnaJ: it exhibited a fitness cost at high growth temperatures (43 °C) and induced morphofunctional alterations in C. elegans. The variant resistant to carvacrol, RV-CAR, had mutations in sseG, flhA, wbaV, lon; this variant not only exhibited significantly higher thermotolerance in both laboratory media and food models but also effectively increased its growth fitness at refrigeration temperatures while retaining its virulence, evidenced by the highest percentage of Smurf phenotype in C. elegans. To address these challenges, we applied a process combining thermal treatment with citral, with the aim of leveraging the sublethal damage caused in RVs by heat treatments in orange juice. This approach achieves enhanced microbial inactivation without having to escalate the intensity of the thermal treatment. The result was particularly encouraging in the case of RV-CAR, the most challenging strain, for which we improved lethality by up to 3 log10 inactivation cycles.

Dynamics of microbiome and resistome in a poultry burger processing line.

Traditionally, surveillance programs for food products and food processing environments have focused on targeted pathogens and resistance genes. Recent advances in high throughput sequencing allow for more comprehensive and untargeted monitoring. This study assessed the microbiome and resistome in a poultry burger processing line using culturing techniques and whole metagenomic sequencing (WMS). Samples included meat, burgers, and expired burgers, and different work surfaces. Microbiome analysis revealed spoilage microorganisms as the main microbiota, with substantial shifts observed during the shelf-life period. Core microbiota of meat and burgers included Pseudomonas spp., Psychrobacter spp., Shewanella spp. and Brochothrix spp., while expired burgers were dominated by Latilactobacillus spp. and Leuconostoc spp. Cleaning and disinfection (C&D) procedures altered the microbial composition of work surfaces, which still harbored Hafnia spp. and Acinetobacter spp. after C&D. Resistome analysis showed a low overall abundance of resistance genes, suggesting that effective interventions during processing may mitigate their transmission. However, biocide resistance genes were frequently found, indicating potential biofilm formation or inefficient C&D protocols. This study demonstrates the utility of combining culturing techniques and WMS for comprehensive of the microbiome and resistome characterization in food processing lines.

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.

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.

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.

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.

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.

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.

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.