Anti-biofilm mechanisms of action of essential oils by targeting genes involved in quorum sensing, motility, adhesion, and virulence: A review.

Biofilms are a critical factor for food safety, causing important economic losses. Among the novel strategies for controlling biofilms, essential oils (EOs) can represent an environmentally friendly approach, able to act both on early and mature stages of biofilm formation. This review reports the anti-biofilm mechanisms of action of EOs against five pathogenic bacterial species known for their biofilm-forming ability. These mechanisms include disturbing the expression of genes related to quorum sensing (QS), motility, adhesion, and virulence. Biofilms and QS are interconnected processes, and EOs interfere with the communication system (e.g. regulating the expression of agrBDCA, luxR, luxS, and pqsA genes), thus influencing biofilm formation. In addition, QS is an important mechanism that regulates gene expression related to bacterial survival, virulence, and pathogenicity. Similarly, EOs also influence the expression of many virulence genes. Moreover, EOs exert their effects modulating the genes associated with bacterial adhesion and motility, for example those involved in curli (csg), fimbriae (fim, lpf), and flagella (fla, fli, flh, and mot) production, as well as the ica genes responsible for synthetizing polysaccharide intercellular adhesin. This review provides a comprehensive framework on the topic for a better understanding of EOs biofilm mechanisms of action.

Challenges and Opportunities in the Sustainable Improvement of Carrot Production.

From an agricultural perspective, carrots are a significant tap root vegetable crop in the Apiaceae family because of their nutritional value, health advantages, and economic importance. The edible part of a carrot, known as the storage root, contains various beneficial compounds, such as carotenoids, anthocyanins, dietary fiber, vitamins, and other nutrients. It has a crucial role in human nutrition as a significant vegetable and raw material in the nutraceutical, food, and pharmaceutical industries. The cultivation of carrot fields is susceptible to a wide range of biotic and abiotic hazards, which can significantly damage the plants' health and decrease yield and quality. Scientific research mostly focuses on important biotic stressors, including pests, such as nematodes and carrot flies, as well as diseases, such as cavity spots, crown or cottony rot, black rot, and leaf blight, caused by bacteria, fungi, and oomycetes. The emerging challenges in the field include gaining a comprehensive understanding of the interaction between hosts and pathogens in the carrot-pathogen system, identifying the elements that contribute to disease development, expanding knowledge of systemic treatments, exploring host resistance mechanisms, developing integrated control programs, and enhancing resistance through breeding approaches. In fact, the primary carrot-growing regions in tropical and subtropical climates are experiencing abiotic pressures, such as drought, salinity, and heat stress, which limit carrot production. This review provides an extensive, up-to-date overview of the literature on biotic and abiotic factors for enhanced and sustainable carrot production, considering the use of different technologies for the shelf-life extension of carrots. Therefore, it addresses the current issues in the carrot production chain, opening new perspectives for the exploration of carrots both as a food commodity and as a source of natural compounds.

Antilisterial Effectiveness of Origanum vulgare var. hirtum and Coridothymus capitatus Essential Oils and Hydrolates Alone and in Combination.

The antimicrobial activity of Origanum vulgare var. hirtum (O) and Coridothymus capitatus (C) essential oils (EOs) and hydrolates (HYs) of the same botanical species was evaluated on sixteen L. monocytogenes strains from food and clinical origins. The antimicrobial activity was assessed by Minimum Inhibitory Concentration (MIC) determination, viable cell enumeration over time up to 60 min, and evaluation of the cellular damage through Confocal Laser Scanning Microscope (CLSM) analysis. EOs exhibited antimicrobial activity with MIC values ranging from 0.3125 to 10 µL/mL. In contrast, HYs demonstrated antimicrobial effectiveness at higher concentrations (125-500 µL/mL). The effect of HYs was rapid after the contact with the cells, and the cell count reduction over 60 min of HY treatment was about 1.2-1.7 Log CFU/mL. L. monocytogenes cells were stressed by HY treatment, and red cell aggregates were revealed through CLSM observation. Moreover, the combinations of EOs and HYs had an additive antilisterial effect in most cases and allowed the concentration of use to be reduced, while maintaining or improving the antimicrobial effectiveness. The combined use of EOs and HYs can offer novel opportunities for applications, thereby enhancing the antimicrobial effectiveness and diminishing the concentration of use. This provides the added benefit of reducing toxicity and mitigating any undesirable sensory effects.

In Vivo Evaluation of the Potential of Thyme and Lemon Hydrolates as Processing Aids to Reduce Norovirus Concentration during Oyster Depuration.

In this study, we evaluated the use of hydrolates, co-products of essential oil distillation, as processing aids to improve the depuration process of Pacific oysters (Crassostrea gigas) as a post-harvest method aimed at reducing the norovirus (NoV) viral load. Live oysters were kept in water to which hydrolates of Thymus serpyllum and Citrus limon at 1% were added for 24 h. The concentration of NoV was quantified using the ISO 15216-1 quantitative real-time RT-PCR method in the oyster digestive tissue both before and after the treatment. The results showed a significant reduction of 0.2 log in the NoV GII concentration after 24 h of treatment with 1% C. limon hydrolate. Conversely, treatment with T. serpyllum did not appear to reduce the concentration of NoV compared to the control. Additionally, a sensory analysis was conducted through a blind survey comparing untreated and treated oysters. No changes in the sensory and physical characteristics of the oysters were observed, except for a decrease in the marine flavour intensity, which was positively perceived by consumers. These results indicate that the addition of hydrolates of C. limon at 1% during depuration might represent a promising processing aid for enhancing both the safety and acceptability of live oysters.

A comprehensive investigation of protein expression profiles in L. monocytogenes exposed to thermal abuse, mild acid, and salt stress conditions.

Preventing L. monocytogenes infection is crucial for food safety, considering its widespread presence in the environment and its association with contaminated RTE foods. The pathogen's ability to persist under adverse conditions, for example, in food processing facilities, is linked to virulence and resistance mechanisms, including biofilm formation. In this study, the protein expression patterns of two L. monocytogenes 1/2a strains, grown under environmental stressors (mild acidic pH, thermal abuse, and high concentration of NaCl), were investigated. Protein identification and prediction were performed by nLC-ESI-MS/MS and nine different bioinformatic software programs, respectively. Gene enrichment analysis was carried out by STRING v11.05. A total of 1,215 proteins were identified, of which 335 were non-cytosolic proteins and 265 were immunogenic proteins. Proteomic analysis revealed differences in protein expression between L. monocytogenes strains in stressful conditions. The two strains exhibited unique protein expression profiles linked to stress response, virulence, and pathogenesis. Studying the proteomic profiles of such microorganisms provides information about adaptation and potential treatments, highlighting their genetic diversity and demonstrating the utility of bioinformatics and proteomics for a broader analysis of pathogens.

Detection and Control of Foodborne Pathogens.

The globalization of food trade and the emergence of disease outbreaks involving several foodborne pathogens and foods has focused the attention of both the research community and consumers on food safety [...].

Stress Adaptation Responses of a Listeria monocytogenes 1/2a Strain via Proteome Profiling.

Listeria monocytogenes is a foodborne pathogen that is ubiquitous and largely distributed in food manufacturing environments. It is responsible for listeriosis, a disease that can lead to significant morbidity and fatality in immunocompromised patients, pregnant women, and newborns. Few reports have been published about proteome adaptation when L. monocytogenes is cultivated in stress conditions. In this study, we applied one-dimensional electrophoresis and 2D-PAGE combined with tandem mass spectrometry to evaluate proteome profiling in the following conditions: mild acid, low temperature, and high NaCl concentration. The total proteome was analyzed, also considering the case of normal growth-supporting conditions. A total of 1,160 proteins were identified and those related to pathogenesis and stress response pathways were analyzed. The proteins involved in the expression of virulent pathways when L. monocytogenes ST7 strain was grown under different stress conditions were described. Certain proteins, particularly those involved in the pathogenesis pathway, such as Listeriolysin regulatory protein and Internalin A, were only found when the strain was grown under specific stress conditions. Studying how L. monocytogenes adapts to stress can help to control its growth in food, reducing the risk for consumers.

Effectiveness of essential oils against dual-species biofilm of Listeria monocytogenes and Pseudomonas fluorescens in a Ricotta-based model system.

Biofilms represent an evolutionary form of life, which translates from life in free-living cells to a community lifestyle. In natural habitats, biofilms are a multispecies complex, where synergies or antagonisms can be established. For example, Listeria monocytogenes and Pseudomonas fluorescens are associated with a dual-species biofilm that is widespread in dairy plants. In food plants, multiple strategies are devised to control biofilms, including natural compounds such as essential oils (EOs). In this respect, this study evaluated the effectiveness of Thymbra capitata (L.) Cav. (TEO) and Cinnamomum zeylanicum (CEO) against a dual-species biofilm of L. monocytogenes and P. fluorescens, mimicking dairy process conditions. Based on Minimum Inhibitory Concentrations results, the EOs concentration (10 µL/mL) was chosen for the antibiofilm assay at 12°C on polystyrene (PS), and stainless-steel surfaces for 168 h, using a Ricotta-based model system as culture medium. Biofilm biomass was assessed by crystal violet staining, and the planktonic and sessile cells were quantified in terms of Log CFU/cm2. Results showed that CEO displayed the greatest antibiofilm activity, reducing significantly (P<0.05) P. fluorescens and L. monocytogenes sessile cells of about 2.5 and 2.8 Log CFU/cm2 after 72 h, respectively. However, L. monocytogenes gained the protection of P. fluorescens, evading CEO treatment and showing a minimal sessile cell reduction of 0.7 Log CFU/cm2 after 72 h. Considering the outcome of this study, CEO might have promising perspectives for applications in dairy facilities.

Effect of membrane depolarization against Aspergillus niger GM31 resistant by ultra nanoclusters characterized by Ag2+ and Ag3+ oxidation state.

To date, the impossibility of treating resistant forms of bacteria and fungi (AMR) with traditional drugs is a cause for global alarm. We have made the green synthesis of Argirium silver ultra nanoclusters (Argirium-SUNCs) very effective against resistant bacteria (< 1 ppm) and mature biofilm (0.6 ppm). In vitro and preclinical tests indicate that SUNCs are approximately 10 times less toxic in human cells than bacteria. Unique chemical-physical characteristics such as particle size < 2 nm, a core composed of Ag0, and a shell of Ag +, Ag2+ , Ag3+ never observed before in stable form in ultra pure water, explain their remarkable redox properties Otto Cars (Lancet Glob. Health 9:6, 2021). Here we show that Argirium-SUNCs have strong antimicrobial properties also against resistant Aspergillus niger GM31 mycelia and spore inactivation (0.6 ppm). The membrane depolarization is a primary target leading to cell death as already observed in bacteria. Being effective against both bacteria and fungi Argirium-SUNCs represent a completely different tool for the treatment of infectious diseases.

The Multifaceted Relationship between the COVID-19 Pandemic and the Food System.

The SARS-CoV-2 pandemic is being questioned for its possible food transmission, due to several reports of the virus on food, outbreaks developed in food companies, as well as its origins linked to the wet market of Wuhan, China. The purpose of this review is to analyze the scientific evidence gathered so far on the relationship between food and the pandemic, considering all aspects of the food system that can be involved. The collected data indicate that there is no evidence that foods represent a risk for the transmission of SARS-CoV-2. In fact, even if the virus can persist on food surfaces, there are currently no proven cases of infection from food. Moreover, the pandemic showed to have deeply influenced the eating habits of consumers and their purchasing methods, but also to have enhanced food waste and poverty. Another important finding is the role of meat processing plants as suitable environments for the onset of outbreaks. Lessons learned from the pandemic include the correct management of spaces, food hygiene education for both food workers and common people, the enhancement of alternative commercial channels, the reorganization of food activities, in particular wet markets, and intensive farming, following correct hygiene practices. All these outcomes lead to another crucial lesson, which is the importance of the resilience of the food system. These lessons should be assimilated to deal with the present pandemic and possible future emergencies. Future research directions include further investigation of the factors linked to the food system that can favor the emergence of viruses, and of innovative technologies that can reduce viral transmission.

Immunoproteome profiling of Listeria monocytogenes under mild acid and salt stress conditions.

Listeria monocytogenes is one of the main foodborne pathogens worldwide. Although its response to stress conditions has been extensively studied, it is still present in the food processing environments and is a concern for consumers. To investigate how this microorganism adapts its proteome in mild stress conditions, a combined proteomics and bioinformatics approach was used to characterize the immunogenic protein profile of a sequence type 7 (ST7) strain that caused severe listeriosis outbreaks in central Italy. Extracted proteins were analyzed by immunoblotting using positive sera against L. monocytogenes and nLC-ESI-MS/MS, and all data were examined by five software to predict subcellular localization (SCL). A total of 226 proteins were extracted from the bands of interest, 58 of which were classified as potential immunogenic antigens. Compared to control cells grown under optimal conditions, six proteins, some of which under-described, were expressed under mild acid and salt stress conditions and/or at 12°C. In particular, adaptation and shaping of the proteome mainly involved cell motility at 12°C without acid and salt stress, whereas the combination of the same temperature with mild acid and salt stress induced a response concerning carbohydrate metabolism, oxidative stress, and DNA repair. Raw data are available via ProteomeXchange with identifier PXD033519.

An overview of the natural antimicrobial alternatives for sheep meat preservation.

Sheep meat is consumed and appreciated all over the world for its nutritional value and flavor. However, this meat is very perishable and easily subjected to the action of both spoilage and pathogenic microorganisms. For this reason, in combination with cold storage, effective preservation techniques are required. There is increasing interest in the application of natural antimicrobials, such as essential oils, extracts, spices, and by-products of the food industry. This review analyses the studies on natural antimicrobials in sheep meat and sheep meat products and gathers evidence about the encouraging results achieved on the reduction and/or elimination of spoilage and pathogenic microorganisms. The use of these natural antimicrobial alternatives might open up important perspectives for industrial application, considering that this specific meat is often traded over long distances. In fact, on the basis of scientific literature, natural antimicrobials can be considered a sustainable and affordable alternative to extend the shelf life of sheep meat and guarantee its safety, although many factors need to be further investigated, such as the sensory impact, potential toxicity, and economic aspects. For all these issues, investigated in some of the studies reviewed here, it is fundamental to obtain the antimicrobial effect with the minimum amount of effective substance to avoid sensory modifications, toxic effects, and unbearable costs. This study sets foundations for the possible direction of future studies, which will contribute to identify effective solutions for industrial applications of natural antimicrobials in the sheep meat industry.

Unraveling the Antimicrobial Effectiveness of Coridothymus capitatus Hydrolate against Listeria monocytogenes in Environmental Conditions Encountered in Foods: An In Vitro Study.

The increased resistance of bacteria to antimicrobials, as well as the growing interest in innovative and sustainable alternatives to traditional food additives, are driving research towards the use of natural food preservatives. Among these, hydrolates (HYs) have gained attention as "mild" alternatives to conventional antimicrobial compounds. In this study, the response of L. monocytogenes ATCC 7644 exposed to increasing concentrations of Coridothymus capitatus HY (CHY) for 1 h at 37 °C was evaluated by means of Phenotype Microarray, modelling the kinetic data obtained by inoculating control and treated cells into GEN III microplates, after CHY removal. The results revealed differences concerning the growth dynamics in environmental conditions commonly encountered in food processing environments (different carbon sources, pH 6.0, pH 5.0, 1-8% NaCl). More specifically, for treated cells, the lag phase was extended, the growth rate was slowed down and, in most cases, the maximum concentration was diminished, suggesting the persistence of stress even after CHY removal. Confocal Laser Scanner Microscopy evidenced a diffuse aggregation and suffering of the treated cells, as a response to the stress encountered. In conclusion, the treatment with HY caused a stressing effect that persisted after its removal. The results suggest the potential of CHY application to control L. monocytogenes in food environments.

A Review of the Botany, Volatile Composition, Biochemical and Molecular Aspects, and Traditional Uses of Laurus nobilis.

Laurus nobilis L. is an aromatic medicinal plant widely cultivated in many world regions. L. nobilis has been increasingly acknowledged over the years as it provides an essential contribution to the food and pharmaceutical industries and cultural integrity. The commercial value of this species derives from its essential oil, whose application might be extended to various industries. The chemical composition of the essential oil depends on environmental conditions, location, and season during which the plants are collected, drying methods, extraction, and analytical conditions. The characterization and chemotyping of L. nobilis essential oil are extremely important because the changes in composition can affect biological activities. Several aspects of the plant's secondary metabolism, particularly volatile production in L. nobilis, are still unknown. However, understanding the molecular basis of flavor and aroma production is not an easy task to accomplish. Nevertheless, the time-limited efforts for conservation and the unavailability of knowledge about genetic diversity are probably the major reasons for the lack of breeding programs in L. nobilis. The present review gathers the scientific evidence on the research carried out on Laurus nobilis L., considering its cultivation, volatile composition, biochemical and molecular aspects, and antioxidant and antimicrobial activities.

Advances in understanding the enzymatic potential and production of ochratoxin A of filamentous fungi isolated from cocoa fermented beans.

In this work, we determined the degree of filamentous fungi contamination in cocoa fermented seeds and the fungal potential to produce enzymes that could contribute to the cocoa quality. Internal transcribed spacer amplicon sequencing (ITS) showed 11 different species with Byssochlamys spectabilis, Aspergillus niger, Aspergillus flavus, Aspergillus carbonarius, Circinella muscae and Penicillium citrinum as the most abundant species. Our results evidenced intra and inter-species differences in the enzymes production. Cellulases, ß-glucosidase, ornithine decarboxylase and phenylalanine decarboxylase were the most diffused enzymes expressed in the 53 strains here studied. Moreover, A. niger (6/12), A. carbonarius (2/3), and P. citrinum (3/3) showed high pectinolytic activity. Remarkable was the amino decarboxylase activity of P. citrinum, and A. flavus strains. For the first time we reported the presence of B. spectabilis in cocoa fermented beans, which could play an important role in the biogenic amines formation. In addition, we explored the capability of the Aspergillus section Nigri strains, to produce ochratoxin A (OTA) in a cacao model system (CPMS) and in malt extract medium (MEL). We observed that CPMS, but not MEL, stimulated the OTA production in 6 out 15 strains of Aspergillus section Nigri, reaching values ranging between 1.70 and 4995 µg OTA kg-1 dry mycelium.

Biogenic Amines in Meat and Meat Products: A Review of the Science and Future Perspectives.

Biogenic amines (BAs) can be found in a wide range of meat and meat products, where they are important as an index for product stability and quality, but also for their impact on public health. This review analyzes the scientific evidence gathered so far on the presence and role of biogenic amines in meat and meat products, also considering the effect of technological conditions on BAs accumulation or decrease. The data provided can be useful for developing solutions to control BAs formation during the shelf-life, for example by novel starters for dry cured products, as well as by packaging technologies and materials for fresh meats. Further research, whose trends are reviewed in this paper, will fill the knowledge gaps, and allow us to protect such perishable products along the distribution chain and in the home environment.

Effectiveness and mechanisms of essential oils for biofilm control on food-contact surfaces: An updated review.

Microbial biofilms represent a constant source of contamination in the food industry, being also a real threat for human health. In fact, most of biofilm-producing bacteria are becoming resistant to sanitizers, thus arousing the interest in natural alternatives to prevent biofilm formation on foods and food-contact surfaces. In particular, studies on biofilm control by essential oils (EOs) application are increasing, being EOs characterized by unique mixtures of compounds able to impair the mechanisms of biofilm development. This review reports the anti-biofilm properties of EOs in bacterial biofilm control (inhibition, removal and prevention of biofilm dispersion) on food-contact surfaces. The relationship between EOs effect and composition, concentration, involved bacteria, and surfaces is discussed, and the possible sites of action are also elucidated. The findings prove the high biofilm controlling capability of EOs through the regulation of genes and proteins implicated in motility, Quorum Sensing and exopolysaccharides (EPS) matrix. Moreover, incorporation in nanosized delivery systems, formulation of blends and combination of EOs with other strategies can increase their anti-biofilm activity. This review provides an overview of the current knowledge of the EOs effectiveness in controlling bacterial biofilm on food-contact surfaces, providing valuable information for improving EOs use as sanitizers in food industries.

Thymus vulgaris L. essential oils from Emilia Romagna Apennines (Italy): phytochemical composition and antimicrobial activity on food-borne pathogens.

Between Modena and Bologna Apennines (Italy), several agricultural farms have recently been dedicated to the cultivation of autochthonous aromatic plants as primary cultivation or to complement other crops. In this study, the chemical composition of Thymus vulgaris L. essential oils (EOs) from this region was evaluated by means of gas-chromatographic analysis. Three different mulching techniques, in particular, soil coverage with grass, mulch with plastic film and with straw were investigated. The results highlighted that mulching techniques influenced the composition of the analysed EOs. All the EOs exerted good antimicrobial activity against clinical and food strains of the pathogen Listeria monocytogenes, with differences related to the composition. The EO obtained from plants covered with grass showed the best results, having MIC ≤ 2.5 µL/mL, and being able to inhibit also antibiotic-resistant strains, thus confirming that soil coverage with grass influences the composition and also the biological activity of Thymus vulgaris EO.

Effectiveness of selected essential oils and one hydrolate to prevent and remove Listeria monocytogenes biofilms on polystyrene and stainless steel food-contact surfaces.

AIMS: This study aimed to evaluate the effectiveness of selected essential oils (EOs) and hydrolates (Hs) against Listeria monocytogenes biofilms on polystyrene (PS) and stainless steel (SS) surfaces. METHODS AND RESULTS: Among others, Origanum hirtum EO, Corydothymus capitatus EO and Citrus aurantium H were selected to treat L. monocytogenes biofilms during and after biofilm formation. Sub-minimum inhibitory concentrations (MICs) of C. capitatus EO (0.31 µl/ml) showed the highest inhibiting effect against biofilm formation on PS, while on SS no significant differences between the EOs were observed (43.7%-88.7% inhibition). Overall, the tested biosanitizers showed limited activity as biofilm removal agents. Although generally less effective, C. aurantium H exhibited good biofilm inhibition performance at 62.5 µl/ml, particularly on PS. Confocal laser scanning microscopy proved that sub-MICs of the biosanitizers drastically changed L. monocytogenes biofilm architecture, with bacterial cells elongation in the presence of C. capitatus EO. CONCLUSIONS: Our findings suggest that the tested EOs and H are able to control Listeria biofilms, particularly preventing biofilm formation on both materials. Considering its mild aroma and hydrophilicity, the H exhibited promising perspectives of application. SIGNIFICANCE AND IMPACT OF STUDY: This study raises the possibility of applying EOs and Hs to control biofilms on different surfaces in the food industry.

Effect of the Distillation Time on the Chemical Composition, Antioxidant Potential and Antimicrobial Activity of Essential Oils from Different Cannabis sativa L. Cultivars.

Within the unavoidable variability of various origins in the characteristics of essential oils, the aim of this study was to evaluate the effect of the distillation time on the chemical composition and biological activity of Cannabis sativa essential oils (EOs). The dry inflorescences came from Carmagnola, Kompolti, Futura 75, Gran Sasso Kush and Carmagnola Lemon varieties from Abruzzo region (Central Italy), the last two being new cultivar here described for the first time. EOs were collected at 2 h and 4 h of distillation; GC/MS technique was applied to characterize their volatile fraction. The EOs were evaluated for total polyphenol content (TPC), antioxidant capacity (AOC) and antimicrobial activity against food-borne pathogens and spoilage bacteria. The time of distillation particularly influenced EOs chemical composition, extracting more or less terpenic components, but generally enriching with minor sesquiterpenes and cannabidiol. A logical response in ratio of time was observed for antioxidant potential, being the essential oils at 4 h of distillation more active than those distilled for 2 h, and particularly Futura 75. Conversely, except for Futura 75, the effect of time on the antimicrobial activity was variable and requires further investigations; nevertheless, the inhibitory activity of all EOs against Pseudomonas fluorescens P34 was an interesting result.