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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., (© 2021 Society for Applied Microbiology.)

Among pathogens, L. monocytogenes has the capability to persist on Food Processing Environment (FPE), first of all posing safety issues, then economic impact on productivity. The aim of this work was to determine the influence of biofilm forming-ability and molecular features on the persistence of 19 Listeria monocytogenes isolates obtained from FPE, raw and processed products of a cold-smoked salmon processing plant. To verify the phenotypic and genomic correlations among the isolates, different analyses were employed: serotyping, Clonal Complex (CC), core genome Multi-Locus Sequence Typing (cgMLST) and Single Nucleotide Polymorphisms (SNPs) clustering, and evaluation of the presence of virulence- and persistence-associated genes. From our results, the biofilm formation was significantly higher (*P < 0.05) at 37 °C, compared to 30 and 12 °C, suggesting a temperature-dependent behaviour. Moreover, the biofilm-forming ability showed a strain-specific trend, not correlated with CC or with strains persistence. Instead, the presence of internalin (inL), Stress Survival Islet (SSI) and resistance to erythromycin (ermC) genes was correlated with the ability to produce biofilms. Our data demonstrate that the genetic profile influences the adhesion capacity and persistence of L. monocytogenes in food processing plants and could be the result of environmental adaptation in response to the external selective pressure., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Understanding the bacterial behavior in response to sublethal stresses is crucial for the optimization of antimicrobial treatments, particularly in food processing. However, the data regarding the effect of sublethal concentrations of essential oils (EOs) on microbial cells are still scarce. In this study, we evaluated the response of Listeria monocytogenes ATCC 7644 to the sublethal concentration of 1.25 μL/mL Origanum vulgare L. EO at 30 °C for 1 h, by means of Phenotype Microarray, modelling the kinetic data obtained by inoculating the control and treated cells into GEN III microplates (Biolog Inc.) for 96 h after EO removal. The results showed important differences regarding the growth dynamics in the presence of carbohydrates and environmental conditions often encountered in food products and that normally sustain the growth of the pathogen (i.e. pH 6.0, pH 5.0, NaCl 1–8%, presence of lactic acid and sodium lactate). In detail, the lag phase of the treated cells was extended, the growth rate was slowed down and the maximum concentration was often significantly reduced. Interestingly, the susceptibility to antimicrobials such as vancomycin, lincomycin and rifamycin was restored after EO application and removal, suggesting a potential role in combating antibiotic resistance. Confocal laser scanner microscopy showed a diffuse aggregation of the treated cells, as a response to the stress encountered. In conclusion, a single exposure to a sublethal concentration of O. vulgare EO causes a stressing effect that persists after its removal. This outcome should be better investigated for potential applications both in food production, to combine low EO concentrations with other hurdles to guarantee food safety, and in medicine, to restore the sensitivity to antibiotics. • L. monocytogenes was exposed to a sublethal quantity of O. vulgare essential oil (OEO). • The single exposure to OEO changed growth dynamics in treated cells. • Restoration of sensitivity to vancomycin, lincomycin, rifamycin was observed. • The stress caused by OEO determined cells aggregation. • Sublethal OEO concentration causes a stressing effect persisting after OEO removal. [ABSTRACT FROM AUTHOR]

In dairy processing environments, many bacterial species adhere and form biofilms on surfaces and equipment, leading to foodborne illness and food spoilage. Among them, Listeria monocytogenes and Pseudomonas spp. could be present in mixed-species biofilms. This study aimed to evaluate the interactions between L. monocytogenes and P. fluorescens in biofilms simulating dairy processing conditions, as well as the capability of P. fluorescens in co-culture to produce the blue pigment in a Ricotta-based model system. The biofilm-forming capability of single- and mixed-cultures was evaluated on polystyrene (PS) and stainless steel (SS) surfaces at 12 °C for 168 h. The biofilm biomass was measured, the planktonic and sessile cells and the carbohydrates in biofilms were quantified. The biofilms were also observed through Confocal Laser Scanning Microscopy analysis. Results showed that only P. fluorescens was able to form biofilms on PS. Moreover, in dual-species biofilms at the end of the incubation time (168 h at 12 °C), a lower biomass compared to P. fluorescens mono-species was observed on PS. On SS, the biofilm cell population of L. monocytogenes was higher in the dual-species than in mono-species, particularly after 48 h. Carbohydrates quantity in the dual-species system was higher than in mono-species and was revealed also at 168 h. The production of blue pigment by P. fluorescens was revealed both in single- and co-culture after 72 h of incubation (12 °C). This work highlights the interactions between the two species, under the experimental conditions studied in the present research, which can influence biofilm formation (biomass and sessile cells) but not the capability of P. fluorescens to produce blue pigment. [ABSTRACT FROM AUTHOR]

Chitosan coating is a promising method for food preservation. This study aims to fill the data gap regarding the application of chitosan (1% and 2%) on vacuum-packed fresh pork stored at 4 °C for 28 days, with particular attention on the effect on Listeria monocytogenes, inoculated as a cocktail of three strains. Chitosan at both concentrations was able to significantly reduce L. monocytogenes counts by more than 1.5 Log CFU/g with respect to control; moreover, it inhibited the growth of mesophilic bacteria and was particularly effective on lactic acid bacteria and yeasts. The beneficial effects on shelf life were demonstrated by both panel test and pH evolution. In fact, panellists did not detect any sensory difference between samples treated with 1% chitosan and control up to 14 days of storage, while after 28 days, a pungent flesh odour was perceived in control samples only. Interestingly, at aw values of fresh meat (0.984), the chitosan coating exhibited a liquid behaviour, with a dynamic viscosity of 229.4 ± 4.2 mPa/s. Chitosan coating applied on vacuum-packed pork loins contained L. monocytogenes growth and improved the microbiological characteristics of the product, with a beneficial effect on product shelf life. [ABSTRACT FROM AUTHOR]

Ready-to-eat (RTE) vegetables, including fresh-cut ones, are exposed to the risk of the foodborne pathogen Listeria monocytogenes. Nowadays, fresh-cut products are widely consumed because of their convenience. Nevertheless, effective washing treatments are required for their preparation, to maintain freshness and microbiological safety. Currently, natural substances are increasingly demanded as alternatives to traditional synthetic washing agents. Therefore, this study investigates the application of Thymbra capitata hydrolate as washing solution for rocket salad (Eruca sativa) stored at 4 °C for 48 h, to evaluate a potential application both during the production process of fresh-cut vegetables, and also at the domestic level. The hydrolate applied at a concentration of 500 μL/mL for 5 min allowed to reduce L. monocytogenes load of about 1 log CFU/g at each time of analysis, and to determine a significant reduction of microbial groups normally found on rocket salad, particularly total mesophilic and psychrotrophic counts. The treatment showed no negative effects on pH, a w , and colour of the rocket salad. At a sensory level, the differences among control and treated samples were minimally perceived only on the first day of analysis but disappeared over time. Therefore, this alternative washing treatment revealed to counteract L. monocytogenes growth and to help maintaining the quality of rocket salad. Hence, it could be optimized for a concrete application, also considering the enhancement of the antilisterial activity, the sustainability and low cost of the hydrolate, which represents a by-product from the production of essential oils. • Washing rocket salad with T. capitata hydrolate reduced L. monocytogenes count. • The treatment also reduced microbial groups normally found on rocket salad. • pH, a w , and colour were similar for control and treated samples. • Sensorial perception of the hydrolate disappeared over time. • The hydrolate had antibacterial effect not affecting rocket salad acceptability. [ABSTRACT FROM AUTHOR]

The potential of chitosan as a possible booster of the antimicrobial activity of Origanum vulgare EO (OEO) against spoilage bacteria and Listeria monocytogenes was investigated in fresh pork meat. Pork fillets were inoculated with 3 L. monocytogenes strains, dipped either in Origanum vulgare (oregano) Essential Oil (OEO) at 2 and 4%, or in chitosan 1% alone or added with 2 and 4% OEO, then packed under modified atmosphere (70% O 2 , 20% CO 2 , 10% N 2 ) and stored at 4 °C for 15 days. OEO did not reduce L. monocytogenes growth, while 2 Log decrease was obtained after 2 days of storage in treatments with chitosan alone or with OEO, with growth inhibition up to day 15 in samples with chitosan and OEO 4%. When OEO was combined with chitosan, total viable counts and spoilage bacteria were reduced and contained over time, particularly Pseudomonas (2.0 Log CFU/g at day 15) and Brochothrix thermosphacta (undetectable). All the treatments applied extended meat shelf-life with respect to control, whose commercial shelf-life was 10 days. Chitosan treatments enhanced L* and maintained a* values almost stable during storage. Chitosan and OEO singly applied reduced lipid oxidation (0.62–0.75 mg malondialdehyde/Kg meat) compared to control (0.99 mg malondialdehyde/Kg meat). Finally, chitosan treated samples were not recognized with respect to the control, whereas OEO gave bitter taste; chitosan with OEO instead mitigated the effect of OEO addition to meat. Chitosan combined with OEO boosts its antimicrobial activity and shows a potential for application in industrial production of fresh pork in MAP, to achieve shelf-life extension, control of L. monocytogenes growth, stability of color and protective effect from oxidation, with low sensory impact. [ABSTRACT FROM AUTHOR]