Listeria monocytogenes is a major foodborne pathogen that significantly threatens public health and food safety. While Thymus vulgaris essential oil (TV-EO) is widely recognized for its potent antibacterial activity, its specific effects against L. monocytogenes remain unexplored. This study aimed to assess the antilisterial activity of TV-EO using in vitro, in situ, and in silico approaches. The in vitro assessment included disc diffusion method, determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), biofilm inhibition assay, and predictive modeling to assess L. monocytogenes reduction in the presence of TV-EO at 10 °C and 20 °C. In situ approach evaluated the inhibitory effect of TV-EO on L. monocytogenes in minced poultry meat stored at 4 °C. Finally, in silico approach, based on molecular docking, was employed to evaluate the binding affinity of major TV-EO components for β-ketoacyl-ACP synthase II and chorismate synthase, key proteins involved in fatty acid biosynthesis and biofilm formation, respectively. Our finding revealed that TV-EO exhibited strong in vitro antilisterial activity, with inhibitory zones ranging from 51.00 ± 1.00 mm to 55.67 ± 1.15 mm, a MIC value of 0.125 %, and a MBC value of 0.25 %, indicating its bactericidal effect. TV-EO at 0.125 % demonstrated a high capacity to inhibit and eradicate the biofilm, with 100 ± 0.00 % and 91.33 ± 1.23 %, respectively. Predictive modeling, based on the combination of TV-EO and ζ values, revealed that L. monocytogenes inactivation was more pronounced at low temperature. Furthermore, the in-situ approach showed a significant reduction of L. monocytogenes amount, with decreases of 1.068 ± 0.132 log cfu/g, 0.671 ± 0.091 log cfu/g, and 0.317 ± 0.029 log cfu/g at TV-EO concentrations of 1 %, 0.5 %, and 0.25 %, respectively (p < 0.05). In silico analysis indicated that TV-EO components, particularly carvacrol, exhibited high affinity for β-ketoacyl-ACP synthase II and chorismate cynthase, suggesting strong antilisterial and ani-biofilm activity. These findings highlight the antilisterial efficacy of TV-EO, demonstrating its potential as a natural alternative to conventional preservatives for enhancing food preservation and safety.

The persistence of the foodborne pathogen Listeria monocytogenes in food processing facilities may be facilitated by the formation of multi-species biofilms by environmental microbiota. This study aimed to determine whether multi-species biofilm formation results in an increased tolerance of L. monocytogenes in biofilms to the sanitizers benzalkonium chloride (BAC) and peroxyacetic acid (PAA) at concentrations commonly used in food processing facilities. Biofilms composed of microbiota previously shown to co-occur with L. monocytogenes in tree fruit packing facilities (i.e., Pseudomonadaceae, Xanthomonadaceae, Flavobacteriaceae, and Microbacteriaceae) were formed with L. monocytogenes in single- and multi-family assemblages. Multi-family biofilms were exposed to 250 or 500 ppm of PAA, or 200 ppm of BAC to determine the die-off kinetics of L. monocytogenes. Furthermore, the ability of a commercial biofilm remover to disrupt biofilms and inhibit bacteria in the formed single- and multi-family assemblage biofilms was assessed. The die-off kinetics of total bacteria and L. monocytogenes in biofilm assemblages throughout the exposure to a sanitizer was determined using the aerobic plate count and the most probable number methods, respectively. Biofilm assemblages that included Pseudomonadaceae resulted in an increased tolerance of L. monocytogenes to BAC and PAA compared to biofilm assemblages without Pseudomonadaceae. Further, the use of the biofilm remover significantly disrupted biofilms and reduced the concentration of L. monocytogenes in single- and multi-family biofilms by 5 or more logarithmic units. These findings highlight the need to improve the control of biofilm-forming microbiota in food processing facilities to mitigate the persistence of L. monocytogenes.

Listeria monocytogenes-targeting phage (LMC) was isolated from fowl droppings, and its complete genome was analyzed. LMC consists of 42,151 bp with a GC content of 36.58% and contains 59 open reading frames. It has an icosahedral capsid and a long tail. It shows a high antibacterial ability against L. monocytogenes ATCC 19115.

Listeria monocytogenes (Lm) may persist in food processing environments (FPEs) alongside diverse background microflora. While microbial communities in FPEs can influence Lm survival, their role in supporting or suppressing its growth remains unclear. This study aimed to characterize the microflora in floor swabs and air samples collected from a dairy processing facility across three seasons and assess their potential impact on the growth of a Lm test strain previously isolated from a dairy processing environment. A total of 167 environmental isolates, representing 30 bacterial genera, were identified. Pseudomonas was consistently prevalent across all sample types. Seasonal shifts in bacterial genera were observed, with differences in microbial composition and relative abundance between production lines with and without Listeria innocua occurrence. Microflora distribution appeared more influenced by environmental and operational factors than by spatial proximity. Co-culture growth assays revealed no competitive exclusion of the Lm test strain, and no zones of inhibition were observed in antimicrobial assays using cell-free extract and dialyzed cell-free extract from environmental isolates against Lm. These findings suggest that Lm could potentially establish itself within mixed microbial communities in dairy processing environments, emphasizing the complexity of microbial interactions in FPEs and their potential role in Lm persistence.

Background/Objectives:Listeria monocytogenes is a significant foodborne pathogen associated with dairy products, which can pose serious public health risks, particularly for vulnerable populations. This study aimed to assess the prevalence, serotype distribution, and antimicrobial resistance profiles of Listeria monocytogenes isolated from dairy products collected in Romania over a three-year period (2021-2023). To the best of our knowledge, this is the first comprehensive study addressing these issues within the country. Methods: A total of 10,306 dairy samples, including milk, cheeses, ice cream, yogurt, and other dairy-based products, were collected and analyzed using standard microbiological methods. Molecular serotyping was performed to identify the most common serogroups. The antimicrobial susceptibility of the isolates was also conducted. Results: The overall prevalence of Listeria monocytogenes was 0.41% (43/10,306). The most frequently detected serogroup was IVb (74.41%), followed by IIa (23.25%) and IIb (2.32%). Ice cream was the most affected product, followed by fresh telemea made from cow milk. Antimicrobial susceptibility testing revealed higher resistance rates for oxacillin and trimethoprim-sulfamethoxazole (13.95% each), while all isolates were susceptible to ciprofloxacin, levofloxacin, and moxifloxacin. Conclusions: The findings emphasize the need for continuous monitoring of Listeria monocytogenes in dairy products, particularly ice cream and fresh cheeses, due to their high contamination rates. The study's results are valuable for comparative analysis with findings from other countries, helping to establish a broader understanding of Listeria monocytogenes contamination trends and resistance profiles.

Listeria monocytogenes, a gram-positive facultative anaerobic bacterium, demonstrates remarkable adaptability to various environmental stressors in food processing environments. It can survive and grow under extremely challenging environmental conditions such as low pH and temperatures, high salinity, and UV radiation. Its ability to generate biofilms at multiple stages of the food processing chain poses significant food safety issues. This bacterium is known for causing severe listeriosis, making it a major problem in microbiology and food safety. L. monocytogenes relies on motility to explore surfaces, attach, and build biofilms. It comprises actin-based motility, which is used for cell-to-cell propagation inside host tissues, and flagellar-driven motility, which assists in surface colonization and infection spread. Flagellar motility also plays an important function in increasing virulence throughout infection cycles. L. monocytogenes motility is regulated by a complex network of regulatory proteins that govern the expression of motility-associated genes. These proteins directly impact pathogenicity by influencing motility and biofilm formation, as well as an indirect impact via regulatory pathways. Efforts to control L. monocytogenes infections and decrease food safety impact include a variety of procedures. Natural compounds, synthetic agents, nanomaterials, and conjugates have emerged as intriguing options for inhibiting motility, disrupting biofilm formation, and reducing virulence. These strategies focus on vital elements of the L. monocytogenes life cycle and pathophysiology to improve food safety and public health. This review provides a comprehensive discussion of the regulatory mechanisms governing L. monocytogenes motility, emphasizing their role in pathogenicity, and explores potential strategies for attenuating the motility and virulence properties. Understanding these mechanisms is essential for developing targeted therapeutic approaches against L. monocytogenes infections and improving food safety practices.

Listeria monocytogenes poses a significant food safety risk, particularly in ready-to-eat (RTE) products, due to its persistence in food processing environments. This study aimed to assess the significance of L. monocytogenes contamination routes, persistence, and monitoring and control in two Spanish food industries: a fresh pork-cutting industry (Industry A) and an RTE food production industry (Industry B). A total of 698 samples from raw materials, final products, food contact surfaces (FCSs), and non-food contact surfaces (NFCSs) were analyzed using impedanciometry, isolation and identification on chromogenic agars, and molecular typing using serotyping and pulsed-field gel electrophoresis. In Industry A, L. monocytogenes contamination increased from 16.7% in raw materials to 53.3% in final products, with four persistent strains detected mainly on FCSs, pointing to their role in pathogen dissemination. In Industry B, the presence of L. monocytogenes decreased from 21.2% in raw materials to undetectable levels in the final products. Only one persistent strain was identified, mainly on NFCSs. Serotype 1/2a predominated in both environments. These findings emphasize the importance of robust monitoring, including contamination characterization, for L. monocytogenes prevention and control. Strengthening control measures in fresh meat processing and enhancing facility and equipment designs could improve overall hygiene and reduce the persistence of L. monocytogenes.

This study explored virulence genes, antibiotic resistance genes, and mobile genetic elements in 14 Listeria monocytogenes strains from milk and dairy products collected from different regions of Ethiopia. The strains were classified into two Multilocus Sequence Typing sequence types (ST2 and ST45) and further grouped into clonal complexes (CC2) and different cgMLST types. Twenty-nine virulence genes were identified across all 14 strains, with lplA1 detected at higher levels in all strains except SAMN28661660. All L. monocytogenes strains also carried four antibiotic resistance genes (fosX, lin, norB, mprF), contributing to their ability to withstand multiple antimicrobial agents. Notably, no plasmids or mobile genetic elements were detected. Stress resistance genes, including stress survival islet 1 (SSI1_lmo0447), lmo 1800, and lmo1799, were identified in all strains. However, genes encoding for disinfectant resistance were not identified from all strains. LGI-2 was found in all the strains and none of the studied strains harbored LGI-1 and LGI-3. Conserved CRISPR-Cas systems were found in some strains. KEGG pathway analysis revealed that inlA and inlB genes facilitate bacterial internalization through host actin polymerization. Overall, the study provided crucial insights into the genomic features of L. monocytogenes in the Ethiopian dairy chain. It is crucial to establish continuous monitoring of L. monocytogenes in dairy products, improve sanitation, enforce stricter antibiotic usage and food safety regulations, and raise public awareness of associated risks.

Foodborne illnesses caused by microorganisms pose a significant threat to public health. Understanding the survival and persistence of these microorganisms in various food matrices is crucial for developing effective control strategies. This systematic review aims to address the current knowledge gaps related to the duration of survival and persistence of microbial pathogens in food, as well as the impact of external environmental conditions on their viability. A comprehensive search was conducted across major databases, including studies published until 3 June 2024. The PRISMA guidelines were followed to ensure a systematic and transparent approach. Foodborne bacteria, such as Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7, were found to persist for extended durations, ranging from days to over a year. The mean duration of persistence for all of the bacteria was 246 days, whereas the survival duration was 16 days. Bacterial survival and persistence were significantly influenced by temperature, with warmer conditions (>25 °C) generally supporting longer persistence. Relative humidity also played a role, with low-humidity environments (<50% RH) favouring the survival of pathogens like Listeria monocytogenes and Escherichia coli. In contrast, viruses, such as hepatitis A virus and Human norovirus, showed only survival patterns, with average durations of 21 days and temperature being the primary environmental factor influencing their survival. Overall, this review provides evidence that a wide range of microbial pathogens, including Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and the hepatitis A virus, can survive and persist in food for prolonged periods, leading to potential harm. These insights underscore the necessity of stringent food safety measures and continuous monitoring to mitigate the risks posed by these resilient pathogens, contributing to a safer and more secure food supply chain.

The filter-feeding nature of oysters, anthropogenic activities, and increasing agriculture in Delaware compromise the microbial safety of Eastern oysters from local aquaculture farms. From July to October 2023, we evaluated the presence and persistence of eight bacteria in seawater and oysters produced from off-bottom and bottom cultures at Sally Cove, an aquaculture farm within Rehoboth Bay in Delaware. A control site within Sally Cove, which was without oyster cultures, was also included in the study. Seawater temperature, salinity, pH, and dissolved oxygen were measured in situ during sampling. Molecular confirmation with PCR and qPCR showed that Vibrio parahaemolyticus, Shiga-toxin-producing Escherichia coli, Salmonella enterica, Staphylococcus aureus, Pseudomonas aeruginosa, and Clostridium spp. were present and persisted in seawater and oyster samples from both cultures at Sally Cove and in off-bottom and bottom seawater samples from the control site throughout the study. Shigella spp. and Listeria monocytogenes were consistently found in seawater and oyster samples from July to September. However, Shigella spp. was only detected in samples from the bottom cultures, whereas L. monocytogenes was undetectable in all samples from both cultures in October. The observed temperature, salinity, pH, and dissolved oxygen levels across the study period were in the range of 15.30-29.67°C, 29.33-31.87 ppt, 7.25-7.95, and 3.79-8.10 mg/L, respectively, and comparable with the conditions suitable for the growth and survival of these bacteria. These findings suggest that consuming raw oysters from Sally Cove poses contamination risks from several bacteria, especially in the summer months.IMPORTANCEAlthough studies have evaluated bacterial contamination in seawater and oysters within the Delaware Inland Bays and nearby areas, the focus has primarily been on Vibrio species. However, other bacteria have been found in seawater and seafood at various locations and could potentially occur in oysters produced from aquaculture farms within the Delaware Inland Bays. Sally Cove is an oyster aquaculture farm that produces Eastern oysters (Crassostrea virginica) for consumption in Delaware using both off-bottom and bottom culturing methods. The risk of bacterial contamination from consuming raw oysters from this farm is unknown. This paper shows the presence and persistence of several bacteria, including those associated with waste, in seawater and oysters at the farm. The findings can inform consumers about the contamination risks from consuming raw oysters produced at the farm.

Listeria monocytogenes is an intracellular, Gram-positive, non-spore-forming, non-encapsulated, facultative anaerobic, rod-shaped, and psychrotrophic food-borne pathogen that causes the infection, listeriosis, thus it attracts great attention following listeriosis outbreaks, which are often associated with high mortality rates. The prevalence of listeriosis is quite low globally; however, the most recent and deadliest outbreak occurred in South Africa, during which 216 persons lost their lives. L. monocytogenes is endowed with the potential to multiply through a wide range of harsh environmental conditions, forming biofilms on varying surfaces in the food industry, as well as having persistent and antibiotic-resistant cells, which pose a major threat and burden to the ready-to-eat food industry. A more frustrating characteristic of this bacterium is its strain divergence, alongside an increased level of antibiotic resistance registered among the strains of L. monocytogenes recovered from food, humans, and environmental sources, especially to those antibiotics involved in the treatment of human listeriosis. Antibiotic resistance exerted by and among pathogenic food-borne microbes is an ongoing public health menace that continues to be an issue. Against this background, a thorough search into different databases using various search engines was performed, which led to the gathering of salient information that was organised, chronologically, based on Listeria monocytogenes and listeriosis. Altogether, the findings elaborated in this study present up-to date knowledge on different aspects of this pathogen which will improve our understanding of the mystery associated with it and the ways to prevent and control its dissemination through ready-to-eat foods. In addition, constant monitoring of the antibiotic resistance profiles of strains of L. monocytogenes from varying sources detected changes, giving an update on the trend in antibiotic resistance. Overall, monitoring of bacterial contamination serves as the key aspect in the control of the food safety output in the food industry.

Listeriosis is an infection caused by the consumption of food contaminated with Listeria monocytogenes. It leads to febrile gastroenteritis, central nervous system infections, and even death in risk populations. Bacteriophage endolysins selectively kill bacteria hydrolyzing their cell walls and have emerged as a potential tool for listeriosis control. Ply511 is an anti-Listeria endolysin that has activity against all serovars of L. monocytogenes. The yeast Saccharomyces cerevisiae has been used to produce endolysins for biocontrol, but prior efforts relied on plasmids, which can lead to gene loss and include selection markers unsuitable for therapeutic use. Integration of endolysins in its genome has also been previously demonstrated, relying however, on selection markers for selection and maintenance of the modifications. This study explores S. cerevisiae as a generally regarded as safe (GRAS) platform for producing and displaying Ply511 through CRISPR-Cas9 integration, offering a marker-free and stable solution for Listeria biocontrol. Our results demonstrate that the surface display of Ply511 does not lead to bacterial reduction. In contrast, we show that yeast secreting endolysin significantly reduces L. monocytogenes in cells, supernatants, and cell extracts. The strongest effect was observed with concentrated spent supernatant and cell extract, which reduced L. monocytogenes below the lower limit of quantification. Additionally, the spent supernatant exhibited active anti-Listeria activity in milk. This study highlights yeast-secreted endolysins as a promising platform for listeriosis control and demonstrates the yeast secretion of endolysins can be used for the biocontrol of pathogenic bacteria. KEY POINTS: • S. cerevisiae was edited using CRISPR-Cas9 to display or secrete endolysin Ply511. • Cells, supernatants, and extracts of yeast secreting Ply511 act against L. monocytogenes. • Demonstrates the yeast-based delivery of endolysins to control L. monocytogenes.

Owing to concerns about the antimicrobial resistance of agents that can prevent the growth of Listeria monocytogenes in meat, researchers have investigated natural preservatives with antilisterial effects. However, in vivo application of essential oils and plant extracts usually results in reduced antimicrobial activity in meat products when compared to in vitro studies. This study aimed to evaluate the in vivo antimicrobial activity of cinnamon essential oil, pomegranate, and strawberry tree extracts in slices of dry-cured ham and pork loin against L. monocytogenes. Fragments of sterile dry-cured ham were inoculated with 100 μL cinnamon oil 0.5%, pomegranate, or strawberry crude extract. After 10 min, 100 μL of L. monocytogenes serotype 4b (104 colony-forming unit [CFU]/mL) was inoculated, and samples were incubated at 7 °C for 7 d to simulate the processing and storage temperature conditions of dry-cured meat products. L. monocytogenes was detected and quantified. Only strawberry extract presented significant differences (P < 0.05) from the control; thus, it was selected for the assay with 2% and 4% salt-treated pork loin. The strawberry tree extract significantly (P < 0.05) reduced the growth of L. monocytogenes in dry-cured ham. However, it could not reduce L. monocytogenes growth in pork loin, regardless of the salt concentration. This is the first report on the antimicrobial effect of strawberry tree leaf extract against L. monocytogenes in dry-cured ham.

Listeria monocytogenes, a rod-shaped Gram-positive bacterium widely distributed in nature, can contaminate foods and represents a foodborne pathogen of public health significance causing a high mortality rate of 20%-30%. Rapid and reliable identification of foods and food-processing environments contaminated with L. monocytogenes is a crucial step in implementing effective intervention strategies to ensure food safety and limit the transmission of bacteria to humans. This study designed and refined a practical workflow to streamline and accelerate the detection of a low level of L. monocytogenes present in ground beef. The workflow coupled an abbreviated 5 h culture enrichment in PALCAM liquid medium with physical separation (filtration and centrifugation) to preprocess enrichment samples. Specific capture was achieved using magnetic separation with a bacteriophage endolysin-derived cell wall-binding domain in a Hyglos Listeria capture kit. Molecular detection was performed using a MicroSEQ L. monocytogenes RTi-PCR detection kit combined with a nested PCR strategy. Preprocessing of enrichment culture samples using a multi-stage filtration system constructed for the study or commercially available BagFilter Pull-up filter bags, in conjunction with centrifugation, enabled the recovery of ~30 colony-forming units (CFUs) from the enrichment culture of a 25 g ground beef sample artificially contaminated with 1 CFU of L. monocytogenes. Integration of magnetic separation into the workflow for capturing L. monocytogenes cells specifically from preprocessed samples and further cleaning up the samples yielded bacterial counts similar to those obtained by direct plating of preprocessed samples. The RTi-PCR-based molecular detection method integrated into the workflow was capable of detecting pure cultures of L. monocytogenes as low as 12.5 CFUs. Evaluation of the workflow using artificially ground beef demonstrated the consistent detection of L. monocytogenes within an 8 h workday in a 25 g sample unit containing the cell count as low as 2 CFU following a 5 h culture enrichment.

Consuming foods contaminated with the bacterial pathogen Listeria monocytogenes can lead to the development of human listeriosis, a severe and life-threatening foodborne illness. Timely detection of L. monocytogenes present at a low level in foods and food processing environments is a necessary measure to prevent the spread of the Listeria-associated illness. This study designed and evaluated a multi-step workflow for testing L. monocytogenes in artificially contaminated food samples. The workflow was composed of a short 5 h culture enrichment, filtration-based sample preprocessing, magnetic separation, a single-tube nested RTi-PCR, and culture plating. It allowed L. monocytogenes to be detected within 8 h from a 25 g ground beef sample containing the target cells as low as 2 colony-forming units, significantly improving and streamlining the detection methods for this important foodborne pathogen.

Listeria monocytogenes is a potentially severe disease-causing bacteria mainly transmitted through food. This pathogen is of great concern for public health and the food industry in particular. Many countries have implemented thorough regulations, and some have even set 'zero-tolerance' thresholds for particular food products to minimise the risk of L. monocytogenes outbreaks. This emphasises that proper sanitation of food processing plants is of utmost importance. Consequently, in recent years, there has been an increased interest in L. monocytogenes tolerance to disinfectants used in the food industry. Even though many studies are focusing on laboratory quantification of L. monocytogenes tolerance, the possibility of predictive models remains poorly studied. Within this study, we explore the prediction of tolerance and minimum inhibitory concentrations (MIC) using whole genome sequencing (WGS) and machine learning (ML). We used WGS data and MIC values to quaternary ammonium compound (QAC) disinfectants from 1649 L. monocytogenes isolates to train different ML predictors. Our study shows promising results for predicting tolerance to QAC disinfectants using WGS and machine learning. We were able to train high-performing ML classifiers to predict tolerance with balanced accuracy scores up to 0.97 ± 0.02. For the prediction of MIC values, we were able to train ML regressors with mean squared error as low as 0.07 ± 0.02. We also identified several new genes related to cell wall anchor domains, plasmids, and phages, putatively associated with disinfectant tolerance in L. monocytogenes. The findings of this study are a first step towards prediction of L. monocytogenes tolerance to QAC disinfectants used in the food industry. In the future, predictive models might be used to monitor disinfectant tolerance in food production and might support the conceptualisation of more nuanced sanitation programs.

Bacterial persistence, a dormant state that enables microorganisms to survive harsh conditions, is a significant concern in food-industry settings, where traditional antimicrobial treatments often fail to eliminate these resilient cells. This article goes beyond conventional review by compiling critical information aimed at providing practical solutions to combat bacterial persisters in food production environments. This review explores the primary mechanisms behind persister cell formation, including toxin-antitoxin systems, the alarmone guanosine tetraphosphate (ppGpp), stochastic processes (in which persistence occurs as a random event), and the SOS response. Given the serious implications for food safety and quality, the authors also report a range of physical, chemical, and biological methods for targeting and eradicating persister cells. The strategies discussed, whether applied individually or in combination, offer varying levels of availability and applicability within the industry and can serve as a guide for implementing microbial contamination control plans. While significant progress has been achieved, further research is crucial to fully understand the complex mechanisms underlying bacterial persistence in food and to develop effective and targeted strategies for its eradication in food-industry settings. Overall, the translation of these insights into practical applications aims to support the food industry in overcoming this persistent challenge, ensuring safer, more sustainable food production.

Listeria (L.) monocytogenes can survive for extended periods in the food producing environment. Here, biofilms possibly provide a niche for long-term survival due to their protective nature against environmental fluctuations and disinfectants. This study examined the behaviour of a L. monocytogenes ST121 isolate in a multispecies biofilm composed of Pseudomonas (P.) fragi, Brochothrix (B.) thermosphacta, and Carnobacterium (C.) maltaromaticum, previously isolated from a meat processing facility. The composition of the biofilm community and matrix, and transcriptional activity were analysed. L. monocytogenes colonised the multispecies biofilm, accounting for 6.4 % of all total biofilm cells after six hours. Transcriptomic analysis revealed 127 significantly up-regulated L. monocytogenes genes compared to the inoculum, including motility, chemotaxis, iron, and protein transport related genes. When comparing the differentially expressed transcripts within the multispecies biofilm with and without L. monocytogenes, only a cadmium/zinc exporting ATPase gene in C. maltaromaticum was significantly upregulated, while the other 9313 genes in the biofilm community showed no significant differential expression. We further monitored biofilm development over time (6, 24 hours and 7 days). P. fragi remained the dominant species, while L. monocytogenes was able to survive in the multispecies biofilm accounting for 2.4 % of total biofilm cells after 7 days, without any significant changes in its abundance. The presence of L. monocytogenes did neither alter the biofilm community nor its matrix composition (amount of extracellular DNA, carbohydrates, and protein). Our data indicate that L. monocytogenes resides in multispecies biofilms, potentially increasing survival against cleaning and disinfection in food processing environments, supporting persistence.

Nisin resistance development is one of food safety challenges posed by Listeria monocytogenes, an important foodborne pathogen that causes human listeriosis. The GtcA flippase enzyme is functionally crucial in two separate pathways that glycosylate cell envelope wall teichoic acids (WTA) with N-acetylglucosamine (NAG) and lipoteichoic acids (LTA) with galactose, respectively. This study investigated phenotypic roles and molecular mechanisms underlying GtcA involvement in L. monocytogenes nisin resistance. A GtcAA65V mutation was linked with increased nisin resistance in a food processing environment associated L. monocytogenes strain. Examination of nisin stress survival and growth phenotypes among L. monocytogenes gtcA mutants in different genetic backgrounds showed that GtcA function promoted sensitivity and loss of its function through genetic deletion (ΔgtcA) and a natural GtcAA65V mutation increased nisin resistance. Individual contributions of GtcA WTA NAG and LTA galactose glycosylation functions to nisin resistance modulation were examined through nisin sensitivity analysis of genetic deletion mutants and L. monocytogenes strains complemented using functionally altered GtcA mutants. This revealed WTA NAG glycosylation to be the main functional mechanism that determines GtcA dependent nisin phenotypic sensitization. An examination for mechanisms underlying GtcA involvement in nisin sensitivity revealed that the loss of GtcA function induces changes in the cell envelope carbohydrate composition profiles reducing cell surface hydrophobicity. Overall, our results showed that cell envelope WTA NAG glycosylation promotes nisin susceptibility through facilitation of hydrophobic interactions between nisin and the Listeria cell envelope. There may be practical implications from our observations since nisin resistance could be gained in food associated L. monocytogenes strains that develop phage resistance through acquisition of mutations in genes that cause loss of cell envelope WTA NAG modifications.

This article examines the impact of fluid flow dynamics on microbial growth, distribution, and control within food processing systems. Fluid flows, specifically laminar and turbulent flows, significantly influence microbial behaviors, such as biofilm development and microbial adhesion. Laminar flow is highly conducive to biofilm formation and microbial attachment because the flow is smooth and steady. This smooth flow makes it much more difficult to sterilize the surface. Turbulent flow, however, due to its chaotic motion and the shear forces that are present, inhibits microbial growth because it disrupts attachment; however, it also has the potential to contaminate surfaces by dispersing microorganisms. Computational fluid dynamics (CFD) is highlighted as an essential component for food processors to predict fluid movement and enhance numerous fluid-dependent operations, including mixing, cooling, spray drying, and heat transfer. This analysis underscores the significance of fluid dynamics in controlling microbial hazards in food settings, and it discusses some interventions, such as antimicrobial surface treatments and properly designed equipment. Each process step from mixing to cooling, which influences heat transfer and microbial control by ensuring uniform heat distribution and optimizing heat removal, presents unique fluid flow requirements affecting microbial distribution, biofilm formation, and contamination control. Food processors can improve microbial management and enhance product safety by adjusting flow rates, types, and equipment configurations. This article helps provide an understanding of fluid-microbe interactions and offers actionable insights to advance food processing practices, ensuring higher standards of food safety and quality control.

With rising concerns about antimicrobial resistance, the identification of new lead compounds to target multidrug-resistant bacteria is essential. This study employed a fast miniaturized screening to simultaneously cultivate and evaluate about 300 marine strains for biosurfactant and antibacterial activities, leading to the selection of the deep-sea Bacillus halotolerans BCP32. The integration of tandem mass spectrometry molecular networking and bioassay-guided fractionation unveiled this strain as a prolific factory of surfactins and nobilamides. Particularly, 84 nobilamide congeners were identified in the bacterial exometabolome, 71 of them being novel metabolites. Among these, four major compounds were isolated, including the known TL-119 and nobilamide I, as well as the two new nobilamides T1 and S1. TL-119 and nobilamide S1 exhibited potent antibiotic activity against various multidrug-resistant Staphylococcus strains and other Gram-positive pathogens, including the foodborne pathogen Listeria monocytogenes. Finally, in silico analysis of Bacillus halotolerans BCP32 genome revealed nobilamide biosynthesis to be directed by a previously unknown heptamodular nonribosomal peptide synthetase.

Listeria monocytogenes is a Gram-positive bacterium responsible for listeriosis, a serious foodborne disease that can lead to serious health complications. Pregnant women, newborns, the elderly, and patients with weakened immune systems are particularly susceptible to infection. Due to the ability of L. monocytogenes to survive in extreme environmental conditions, such as low temperatures, high salinity, and acidity, this bacterium poses a serious threat to food production plants and is particularly difficult to eliminate from these plants. One of the promising solutions to reduce the presence of this bacterium in food products is bacteriocins as natural control agents. These are substances with antibacterial activity produced by other bacteria, mainly lactic acid bacteria (LAB), which can effectively inhibit the development of pathogens such as L. monocytogenes. The use of bacteriocins in the food industry is beneficial due to their natural origin, specificity of action, and consumer safety. However, the problem of resistance to these substances exists.

This review focuses on the mechanisms of bacteriocin resistance, such as modifications of bacteriocin docking receptors, changes in the structure of the cell wall and membrane, and the occurrence of cross-resistance to different bacteriocins. Genetic factors determining these mechanisms and strategies to cope with the problem of resistance are also presented.

Research on this issue is crucial for developing effective preventive methods that will enable the safe and long-term use of bacteriocins in food production.

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.

Ligilactobacillus salivarius harbors bacteriocin genes in its repA-type megaplasmid, specifically salivaricin P (salP), a class IIb bacteriocin. This study aimed to differentiate 25 salP-positive Lig. salivarius strains isolated from the gastrointestinal tract (GIT) of broilers and laying hens. Results showed that 12 isolates were classified as Type A, with active bacteriocins, while the rest were Type B, with no active bacteriocins. In vitro and in silico characterization of salP bacteriocins revealed narrow-spectrum antibacterial activity against Listeria monocytogenes and Enterococcus faecalis. SalP bacteriocins were predicted as positively charged, hydrophobic, small molecular weight (α, 4.097 kDa; ß, 4.285 kDa) bacteriocins with characteristic GXXXG motif. Investigation of the salP gene cluster based on genomic data revealed that Type B strains lacked the lanT and hlyD genes that encode export proteins dedicated to the modification and extracellular transport of mature salP peptides. However, two Type B strains (B4311 and B5258) showed inhibitory activity against L. monocytogenes ATCC19114. Multiplex PCR analysis and synteny mapping analysis revealed that B4311 and B5258 strains harbored the lanT gene, highlighting the importance of LanT protein in the cleavage of leader peptide and excretion of mature peptides. Further analysis revealed that the resistance of Type B strains to salP was attributable to the presence of a dedicated immunity protein, blurring the evolutionary significance of producing active bacteriocins for competitive advantage. Additionally, the loss of export proteins occurred in a polyphyletic manner, consistent with the genetic plasticity of the repA-type megaplasmid. This suggests that the loss of lanT and hlyD is likely in the presence of limited nutritional competitors. In conclusion, the observed differences in salivaricin production of Lig. salivarius exist independent of isolation host and that Type A and Type B strains can coexist in the same environment. Finally, the functional characterization of active salP allows for a better understanding of its potential to control specific bacteria in human food and animal production.

Microwave electrodeless ultraviolet (MWUV) technology, as an emerging food processing technique, has garnered growing attention in the realm of food science in recent years. Based on different application requirements, MWUV equipment types are categorized as microwave oven reactor, continuous-flow UV-microwave reactor, coaxially driven MWUV reactor, and complete ultraviolet reactor. The luminescence properties of MWUV equipment depend on their filler gas; mercury is commonly used as a filler gas to produce a wavelength at 253.7 nm for food non-thermal sterilization. The microbial sterilization effect of MWUV is primarily attributed to the synergistic action of microwave and ultraviolet (UV): MWUV enhances reactive oxygen species (ROS) production, disrupts the cell membrane structures of bacteria, leads to bacterial endosome leakage, and induces nucleic acid damage. MWUV extends food shelf-life by eliminating microorganisms without significantly altering food quality compared with traditional thermal sterilization methods. Additionally, MWUV, combined with digestion reagents such as HNO3 and H2O2, can effectively enhance the digestion of food samples to detect essential and toxic elements. Studies on MWUV technology hold broad potential in the food industry, with promising implications for food safety and consumer demand for high-quality food. Future research may focus on optimizing the equipment parameters and integrating with other food processing technologies to facilitate further development and application of MWUV.

Pathogenic microbial contamination (bacteria and fungi) in food products during production poses a significant global health risk, leading to food waste, greenhouse gas emissions, and aesthetic and financial losses. Bacteria and fungi, by forming solid biofilms, enhance their resistance to antimicrobial agents, thereby increasing the potential for cross-contamination of food products. Curcumin molecule-mediated photodynamic inactivation (Cur-m-PDI) technology has shown promising results in sterilizing microbial contaminants and their biofilms, significantly contributing to food preservation without compromising quality. Photosensitizers (curcumin) absorb light, leading to a chemical reaction with oxygen and producing reactive oxygen species (ROS) that effectively reduce bacteria, fungi, and biofilms. The mechanism of microorganism inhibition is caused by exposure to ROS generated via the type 1 pathway involving electron transfer (such as O2•-, H2O2, -OH•, and other radicals), the type 2 pathway involving energy transfer (such as 1O2), secondary ROS, and weakening of antioxidant enzymes. The effectiveness of the inactivation of microorganisms is influenced by the concentration of curcumin, light (source type and energy density), oxygen availability, and duration of exposure. This article reviews the mechanism of reducing microbial food contamination and inhibiting their biofilms through Cur-m-PDI. It also highlights future directions, challenges, and considerations related to the effects of ROS in oxidizing food, the toxicity of PDI to living cells and tissues, conditions/types of food products, and the stability and degradation of curcumin.

Photodynamic inactivation is an emerging antimicrobial treatment that can be enhanced by employing exogenous photosensitizers to eradicate foodborne pathogens. This study investigated a novel combinatory strategy to eradicate Listeria monocytogenes using blackthorn fruit peel (BFP) and blue light (BL). Extracts of BFP were characterized in terms of polyphenolic content, individual constituents, and antioxidant and antimicrobial activity. The concentration of phenolic compounds and antioxidant activity were both found to be determinants of antimicrobial activity. It was further speculated that flavonols, predominantly quercetin and rutin, were responsible for the activity of BFP against L. monocytogenes. A combination of BFP and BL resulted in a rapid inactivation of the pathogen by up to 4 log CFU/mL at 58.5 J/cm2, corresponding to 15 min BL illumination. Flow cytometry analysis revealed that the bacterial cells lost activity and suffered extensive membrane damage, exceeding 90% of the population. After photosensitizing L. monocytogenes with the BFP constituents quercetin and rutin, a 1.3-log reduction was observed. When applied together, these compounds could inflict the same damaging effect on cells as they did individually when effects were added. Therefore, the results indicate that BFP represents a natural source of (pro-)photosensitizers, which act additively to create inactivation effects. This study may help identify more effective plant-based photosensitizers to control L. monocytogenes in food-related applications.

Foodborne pathogens are microorganisms that cause illness through contamination, presenting significant risks to public health and food safety. This review explores the metabolites produced by these pathogens, including toxins and secondary metabolites, and their implications for human health, particularly concerning cancer risk. We examine various pathogens such as Salmonella sp., Campylobacter sp., Escherichia coli, and Listeria monocytogenes, detailing the specific metabolites of concern and their carcinogenic mechanisms. This study discusses analytical techniques for detecting these metabolites, such as chromatography, spectrometry, and immunoassays, along with the challenges associated with their detection. This study covers effective control strategies, including food processing techniques, sanitation practices, regulatory measures, and emerging technologies in pathogen control. This manuscript considers the broader public health implications of pathogen metabolites, highlighting the importance of robust health policies, public awareness, and education. This review identifies research gaps and innovative approaches, recommending advancements in detection methods, preventive strategies, and policy improvements to better manage the risks associated with foodborne pathogens and their metabolites.

Listeria meningitis is an infectious disease of the central nervous system caused by Listeria monocytogenes. This bacterium is widely present in the natural environment and can be transmitted through channels such as food and water. Patients usually show symptoms such as fever, headache, and neck stiffness. In severe cases, coma, convulsions, or even death may occur. Traditional diagnostic methods, such as cerebrospinal fluid (CSF) culture and serological tests, have certain limitations. Although CSF culture is the "gold standard" for diagnosis, it is time-consuming and has a relatively low positivity rate. Serological detection may also result in false positive or false negative results. The emergence of metagenomic sequencing (mNGS) technology has led to a significant breakthrough in diagnosing Listeria meningitis, allowing quick and accurate detection of various pathogens in samples.

Here, we present the case of a previously healthy 64-year-old woman diagnosed with Listeria meningitis using mNGS. She was successfully treated with intravenous ampicillin and meropenem, without any complications.

Listeria meningitis must be considered, especially in patients who fail to show improvement with first-line antibiotic treatments. mNGS significantly reduces the diagnosis time, supporting timely treatment of patients.

Listeria monocytogenes biofilm contamination on food contact surfaces is a major concern for the food industry. Nanoparticle encapsulation appears as a novel strategy for food surface disinfection to prevent biofilm formation. Chitosan nanoparticles loaded with nisin and DNase I (DNase-CS-N) have been constructed to exhibit antimicrobial activity against L. monocytogenes. This study aimed to investigate their ability to inhibit L. monocytogenes biofilm formation and eliminate preformed biofilms on food contact surfaces (polystyrene, polyurethane, and stainless steel). DNase-CS-N could decrease 99% and 99.5% biofilm cell numbers at 1/2 MIC and MIC, respectively. At sub-MICs, DNase-CS-N could reduce cell motility (swimming and swarming) and slime production of L. monocytogenes. In terms of effect on biofilm elimination, DNase-CS-N at the concentration of 4 MIC led to 3-4 log reduction in biofilm cells in preformed biofilms, performing higher efficiency compared with other treatments (CSNPs, CS-N). Furthermore, the three-dimensional structure of L. monocytogenes biofilms was severely disrupted after DNase-CS-N treatment, with bacterial cells scattered on the surface. The morphology of biofilm cells was also greatly damaged with wrinkled surfaces, disrupted cell membranes, and leakage of intracellular nucleic acids and proteins. These results indicate the potential applicability of DNase-CS-N for inhibiting and eliminating L. monocytogenes biofilms on food contact surfaces.

This research paper presents the characterization of an enterocin-producing Enterococcus durans MF5 isolate and the determination of the in vitro antilisterial activity of enterocin produced by this isolate, named Ent-MF5. PCR-based screening for bacteriocin biosynthetic genes revealed that E. durans MF5 harbors multiple enterocin-encoding genes (ent A, B, P and X), classified as class II bacteriocins and enterocin-P of Enterococcus faecium (sharing up to 99% similarity at the genetic level). E. durans MF5 is sensitive to eight clinically important antibiotics and does not possess cytolysin activator -cylA, gelatinase -gelE and hyaluronidase -hylA virulence genes. The antilisterial activity of Ent-MF5 was abolished by trypsin, α-chymotrypsin, protease and proteinase-K. Ent-MF5 showed thermal and pH stability. In addition, the activity of Ent-MF5 was unaffected in the presence of various surfactants (1% SDS, Triton X-100, Tween 20, and Tween 80). Ent-MF5 exhibited antimicrobial activity against Listeria monocytogenes, Listeria innocua, Listeria ivanovii and Listeria seeligeri at concentrations as low as 0.13 μg/ml. Ent-MF5 had a bactericidal effect against L. monocytogenes with a significant reduction in surviving cells at concentrations equal to or greater than 0.13 μg/ml. A 75-100% reduction in L. monocytogenes growth and bactericidal effect determined by CFU counts was observed following treatment with Ent-MF5 at 4.47 μg/ml at time points starting at 2 and 4 h, respectively. Ent-MF5 action is associated with Listeria cell membrane damage, as observed by flow cytometry and fluorescence microscopy. Thus, the effective antilisterial activity and stability of Ent-MF5 presents promising perspectives for application as biopreservatives in the food industry.

Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis, a severe foodborne illness that is particularly dangerous during pregnancy. It thrives in diverse environments, including refrigerated conditions and food production facilities, due to its adaptability to varying temperatures, pH levels, and salt concentrations. Its virulence stems from the ability to invade host cells, particularly macrophages and epithelial cells, and avoid, or at least postpone, immune detection by utilizing virulence factors such as internalins, listeriolysin O, and actin assembly-inducing protein. This intracellular motility and biofilm formation make LM a persistent pathogen in food safety and public health. Pregnant women are at a much higher risk of listeriosis, which can result in serious fetal complications such as miscarriage, stillbirth, and preterm labor due to LM's affinity for placental tissues. The vertical transmission of LM from mother to fetus can lead to neonatal listeriosis, which can result in sepsis and meningitis, with high mortality rates if not promptly treated. Early diagnosis and treatment with antibiotics, such as ampicillin or gentamicin, are crucial for maternal and neonatal outcomes.

Listeria monocytogenes (L. monocytogenes), a gram-positive foodborne pathogen that can easily cause listeriosis. It secretes extracellular polymers and forms biofilms that are highly resistant to disinfection methods, such as UV light and germicides, posing risks to food processing equipment and food quality. Dispersion of biofilm is the cycle of its formation in which the bacteria return to planktonic state and become susceptible to antimicrobials, the strategic manipulation of biofilm dispersion is thus heralded as a novel and promising approach for the effective control of biofilm-related infections. Compared to the traditional methods, it is more effective to start with the composition of biofilms, cut off the production of their constituent substances, and genetically reduce the probability of biofilm formation. Meanwhile, the dispersion of bacteria can be supplemented with exogenous substances, making long-term control possible. This paper provides a brief but comprehensive overview of the mechanisms of L. monocytogenes biofilms or cross-contamination and their resistance properties, and facilitates our understanding and control of the prevention and containment of L. monocytogenes biofilm contamination based on the biofilm's active and passive diffusion strategies. This work provides practical guidelines for the food industry to guard against the enduring threat to food safety due to L. monocytogenes biofilms.

Listeria monocytogenes is an important human and animal pathogen able to cause an infection named listeriosis and is mainly transmitted through contaminated food. Among its virulence traits, the ability to form biofilms and to survive in harsh environments stand out and lead to the persistence of L. monocytogenes for long periods in food processing environments. Virulence and biofilm formation are phenotypes regulated by quorum sensing (QS) and, therefore, the control of L. monocytogenes through an anti-QS strategy is promising. This study aimed to identify, by in silico approaches, proteins secreted by lactic acid bacteria (LAB) potentially able to interfere with the agr QS system of L. monocytogenes. The genome mining of Lacticaseibacillus rhamnosus GG and Lactobacillus acidophilus NCFM revealed 151 predicted secreted proteins. Concomitantly, the three-dimensional (3D) structures of AgrB and AgrC proteins of L. monocytogenes were modeled and validated, and their active sites were predicted. Through protein-protein docking and molecular dynamic, Serine-type D-Ala-D-Ala carboxypeptidase and L,D-transpeptidase, potentially secreted by L. rhamnosus GG and L. acidophilus NCFM, respectively, were identified with high affinity to AgrB and AgrC proteins, respectively. By inhibiting the translocation of the cyclic autoinducer peptide (cyclic AIP) via AgrB, and its recognition in the active site of AgrC, these LAB proteins could disrupt L. monocytogenes communication by impairing the agr QS system. The application of the QS inhibitors predicted in this study can emerge as a promising strategy in controlling L. monocytogenes in food processing environment and as an adjunct to antibiotic therapy for the treatment of listeriosis.

Listeria are Gram-negative intracellular foodborne pathogens that can cause invasive infections with high mortality rates. In this work, the antibacterial activity of ten essential oils, infusion extracts, and decoction extracts of some medicinal plants was tested against Listeria monocytogenes and listeria ivanovii strains. The effects of different physical conditions including temperature, pH, sodium chloride, and some organic acids were studied. The results showed that the water extracts gave the maximum bacterial inhibition, while ethanolic extract was inactive against the tested Listeria spp. The antibiotic sensitivity of L. monocytogenes LMG10470 and L. ivanovii LMZ11352 was tested against five antibiotics including imipenem, levofloxacin, amikacin, ampicillin, and amoxicillin. Imipenem was the most effective antibiotic, resulting in inhibition zones of 40 mm and 31 mm for L. monocytogenes and L. ivanovii, respectively. When imipenem mixed with Syzygium aromaticum oil, Salvia officinalis oil, Pimpinella anisum infusion, and Mentha piperita infusion each, the water extract of Moringa oleifera leaves and seeds against LMG10470 and LMZ11352 resulted in broader antibacterial activity. The antimicrobial activity of both Pimpinella anisum and Mentha piperita plant extracts is related to a variety of bioactive compounds indicated by gas chromatography-mass spectrometry analysis of these two plant extracts. These two plant extracts seemed to contain many chemical compounds elucidated by gas chromatography-mass spectrometry (GC-MS) and infrared radiation spectra. These compounds could be classified into different chemical groups such as ethers, heterocyclic compounds, aromatic aldehydes, condensed heterocyclic compounds, ketones, alicyclic compounds, aromatics, esters, herbicides, saturated fatty acids, and unsaturated fatty acids. The use of these natural compounds seems to be a useful technological adjuvant for the control of Listeria spp. in foods.

The foodborne bacterium Listeria monocytogenes (Lm) causes a range of diseases, from mild gastroenteritis to invasive infections that have high fatality rate in vulnerable individuals. Understanding the population genomic structure of invasive Lm is critical to informing public health interventions and infection control policies that will be most effective especially in local and regional communities.

We sequenced the whole draft genomes of 936 Lm isolates from human clinical samples obtained in a two-decade active surveillance program across 58 counties in New York State, USA. Samples came mostly from blood and cerebrospinal fluid. We characterized the phylogenetic relationships, population structure, antimicrobial resistance genes, virulence genes, and mobile genetic elements.

The population is genetically heterogenous, consisting of lineages I-IV, 89 clonal complexes, 200 sequence types, and six known serogroups. In addition to intrinsic antimicrobial resistance genes (fosX, lin, norB, and sul), other resistance genes tetM, tetS, ermG, msrD, and mefA were sparsely distributed in the population. Within each lineage, we identified clusters of isolates with ≤ 20 single nucleotide polymorphisms in the core genome alignment. These clusters may represent isolates that share a most recent common ancestor, e.g., they are derived from the same contamination source or demonstrate evidence of transmission or outbreak. We identified 38 epidemiologically linked clusters of isolates, confirming eight previously reported disease outbreaks and the discovery of cryptic outbreaks and undetected chains of transmission, even in the rarely reported Lm lineage III (ST3171). The presence of animal-associated lineages III and IV may suggest a possible spillover of animal-restricted strains to humans. Many transmissible clones persisted over several years and traversed distant sites across the state.

Our findings revealed the bacterial determinants of invasive listeriosis, driven mainly by the diversity of locally circulating lineages, intrinsic and mobile antimicrobial resistance and virulence genes, and persistence across geographical and temporal scales. Our findings will inform public health efforts to reduce the burden of invasive listeriosis, including the design of food safety measures, source traceback, and outbreak detection.

The use of natural and sustainable additives, that are less aggressive to the environment, is a trend in the food industry. Rhamnolipids (RL) biosurfactants have shown potential for controlling food pathogens however, due to the presence of free carboxyl groups, the pH and ionic strength may influence the properties of such surfactants. In this study, we describe the antimicrobial activity of RL under different pH values and NaCl concentrations, towards both planktonic and biofilms of Listeria monocytogenes. RL were effective at pH 5.0 and the addition of 5 % NaCl improved the bactericidal efficacy for planktonic and sessile cells. The effect of NaCl was more pronounced at pH above 6 showing a significant increase in RL antimicrobial activity. At pH 7.0 planktonic population was eradicated by RL only when salt was present whereas biofilm viability was decreased by 5 log with MBIC varying from > 2500.0 mg/L (RL) to 39.0 mg/L (RL + 5 % NaCl). Larger vesicular and lamellar RL self-assembly structures were predominant when NaCl was present, suggesting their association with the antimicrobial activity observed. The pH and ionic strength of the medium are important parameters to be considered for the development of RL-based strategies to control L. monocytogenes.

The ability of foodborne pathogens to grow in food products increases the associated food safety risks. Listeria monocytogenes (Lm) is a highly adaptable pathogen that can survive and grow under a wide range of environmental circumstances, including otherwise inhibitory conditions, such as restrictive cold temperatures. It can also survive long periods under adverse environmental conditions. This review examines the experimental evidence available for the survival and growth of Lm on fresh vegetables and ready-to-eat vegetable salads. Published data indicate that, depending on certain intrinsic (e.g., nutrient composition) and extrinsic factors (e.g., storage temperature, packaging atmosphere), Lm can survive on and in a wide variety of vegetables and fresh-cut minimally processed vegetable salads. Studies have shown that temperature, modified atmosphere packaging, relative humidity, pH, water activity, background microbiota of vegetables, microbial strain peculiarities, and nutrient type and availability can significantly impact the fate of Lm in vegetables and vegetable salads. The influence of these factors can either promote its growth or decline. For example, some studies have shown that background microbiota inhibit the growth of Lm in vegetables and minimally processed vegetable salads, but others have reported a promoting, neutral, or insignificant effect on the growth of Lm. A review of relevant literature also indicated that the impact of most influencing factors is related to or interacts with other intrinsic or extrinsic factors. This literature synthesis contributes to the body of knowledge on possible strategies for improving food safety measures to minimize the risk of Lm-associated foodborne outbreaks involving vegetables and vegetable salads.

Whole cell microbial biosensors (WCMB) are mostly genetically modified microorganisms used to detect target molecules as indicators of biological and chemical contaminants as well as in the identification of compounds of interest in the food industry. The specificity and sensitivity of these biosensors are achieved through the design of genetic circuits that make use of genetic sequences such as promoters, terminators, genes encoding regulatory proteins or reporter proteins, among others. Despite the advances of WCMBs for their application, significant challenges are faced, such as cell stability, regulatory restrictions, and the need to optimize response times so that they can be a competitive detection tool in the market. This review explores the technological progress, potential and limitations of WCMBs in the food industry, starting by reviewing the operating principles of biosensors. The importance of selecting appropriate chassis cells and the integration of recognition elements and transducers to maximize their effectiveness in the detection of contaminants and compounds of interest in the food industry is highlighted.

Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8+ T cell response, but very few T cell epitopes are known in mice as a common animal infection model for listeriosis. To identify epitopes, we screened for Listeria immunopeptides presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. We mapped more than 6000 mouse self-peptides presented on MHC class I molecules, including 12 high confident Listeria peptides from 12 different bacterial proteins. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to activate CD8+ T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. The epitope showed high biological potency in a prime boost model and can be used as a research tool to probe CD8+ T cell responses in the mouse models of Listeria infection. Together, our results demonstrate the power of immunopeptidomics for bacterial antigen identification.

Due to the reports describing virulent and multidrug resistant enterococci, their use has become a topic of controversy despite most of them being safe and commonly used in traditionally fermented foods worldwide. We have characterized Enterococcus lactis SF68, a probiotic strain approved by the European Food Safety Authority (EFSA) for use in food and feed, and find that it has a remarkable potential in food fermentations. Genome analysis revealed the potential of SF68 to metabolize a multitude of carbohydrates, including lactose and sucrose, which was substantiated experimentally. Bacteriocin biosynthesis clusters were identified and SF68 was found to display a strong inhibitory effect against Listeria monocytogenes. Fermentation-wise, E. lactis SF68 was remarkably like Lactococcus lactis and displayed a clear mixed-acid shift on slowly fermented sugars. SF68 could produce the butter aroma compounds, acetoin and diacetyl, the production of which was enhanced under aerated conditions in a strain deficient in lactate dehydrogenase activity. Overall, E. lactis SF68 was found to be versatile, with a broad carbohydrate utilization capacity, a capacity for producing bacteriocins, and an ability to grow at elevated temperatures. This is key to eliminating pathogenic and spoilage microorganisms that are frequently associated with fermented foods.

(1) Background: L. monocytogenes is a food pathogen of great importance, characterized by a high mortality rate. Quaternary ammonium compounds (QACs), such as benzalkonium chloride (BC), are often used as disinfectants in food processing facilities. The effectiveness of disinfection procedures is crucial to food safety. (2) Methods: A collection of 153 isolates of L. monocytogenes from meat processing industry was analyzed for their sensitivity to BC using the agar diffusion method. Genes of interest were detected with PCR. (3) Results: Genes emrC, bcrABC, and qacH were found in 64 (41.8%), 6 (3.9%), and 1 isolate (0.7%), respectively, and 79 isolates (51.6%) were classified as having reduced sensitivity to BC. A strong correlation between carrying QACs resistance-related genes and phenotype was found (p-value < 0.0001). Among 51 isolates originating from bacon (collected over 13 months), 48 had the emrC gene, which could explain their persistent presence in a processing facility. Isolates with the ilsA gene (from LIPI-3) were significantly (p-value 0.006) less likely to carry QACs resistance-related genes. (4) Conclusions: Reduced sensitivity to QACs is common among L. monocytogenes from the meat processing industry. Persistent presence of these bacteria in a processing facility is presumably caused by emrC-induced QACs resistance.

Listeriosis is a severe disease caused by the foodborne pathogen Listeria monocytogenes, posing a significant risk to vulnerable populations such as the elderly, pregnant women, and newborns. While relatively uncommon, it has a high global mortality rate of 20-30%. Recent research indicates that smaller outbreaks of the more severe, invasive form of the disease occur more frequently than previously thought, despite the overall stable infection rates of L. monocytogenes over the past 10 years. The ability of L. monocytogenes to form biofilm structures on various surfaces in food production environments contributes to its persistence and challenges in eradication, potentially leading to contamination of food and food production facilities. To address these concerns, this review focuses on recent developments in epidemiology, risk evaluations, and molecular mechanisms of L. monocytogenes survival in adverse conditions and environmental adaptation. Additionally, it covers new insights into strain variability, pathogenicity, mutations, and host vulnerability, emphasizing the important events framework that elucidates the biochemical pathways from ingestion to infection. Understanding the adaptation approaches of L. monocytogenes to environmental stress factors is crucial for the development of effective and affordable pathogen control techniques in the food industry, ensuring the safety of food production.

The effect of lactocin AL705, bacteriocin produced by Latilactobacillus (Lat.) curvatus CRL1579 against Listeria biofilms on stainless steel (SS) and polytetrafluoroethylene (PTFE) coupons at 10 °C was investigated. L. monocytogenes FBUNT showed the greatest adhesion on both surfaces associated to the hydrophobicity of cell surface. Partially purified bacteriocin (800 UA/mL) effectively inhibited L. monocytogenes preformed biofilm through displacement strategy, reducing the pathogen by 5.54 ± 0.26 and 4.74 ± 0.05 log cycles at 3 and 6 days, respectively. The bacteriocin-producer decreased the pathogen biofilm by ∼2.84 log cycles. Control and Bac- treated samples reached cell counts of 7.05 ± 0.18 and 6.79 ± 0.06 log CFU/cm2 after 6 days of incubation. Confocal scanning laser microscopy (CLSM) allowed visualizing the inhibitory effect of lactocin AL705 on L. monocytogenes preformed biofilms under static and hydrodynamic flow conditions. A greater effect of the bacteriocin was found at 3 days independently of the surface matrix and pathogen growth conditions at 10 °C. As a more realistic approach, biofilm displacement strategy under continuous flow conditions showed a significant loss of biomass, mean thickness and substratum coverage of pathogen biofilm. These findings highlight the anti-biofilm capacity of lactocin AL705 and their potential application in food industries.

Grapevines (Vitis spp.) produce several valuable polyphenol-type secondary metabolites including various stilbenoids. Although the potential application of stilbenes may offer alternative solutions to food safety or health challenges, only little information is available on their antibacterial activity against foodborne pathogens. In this work, high-performance liquid chromatography was used to analyze the stilbenoid profile of various wild Vitis species, including V. amurensis, V. davidii, V. pentagona, and V. romanetii, selected from the gene bank for grapes at the University of Pécs, Hungary. We found that the stilbene profile of cane extracts is strongly genotype-dependent, showing the predominant presence of ε-viniferin with a wide concentration range ≈ 320-3870 µg/g dry weight. A novel yet simple and efficient extraction procedure was developed and applied for the first time on grape canes, resulting in ε-viniferin-rich crude extracts that were tested against Listeria monocytogenes, an important foodborne pathogen. After 24 h exposure, V. pentagona and V. amurensis crude extracts completely eliminated the bacteria at a minimum bactericidal concentration of 42.3 µg/mL and 39.2 µg/mL of ε-viniferin, respectively. On the other hand, V. romanetii extract with 7.8 µg/mL of ε-viniferin resulted in 4 log reduction in the viable bacterial cells, while V. davidii extract with 1.4 µg/mL of ε-viniferin did not show significant antibacterial activity. These findings indicate that the ε-viniferin content was directly responsible for the antibacterial effect of cane extract. However, pure ε-viniferin (purity > 95%) required a higher concentration (188 µg/mL) to eradicate the bacteria under the same conditions, suggesting the presence of other antibacterial compounds in the cane extracts. Investigating the onset time of the bactericidal action was conducted through a kinetic experiment, and results showed that the reduction in living bacterial number started after 2 h; however, the bactericidal action demanded 24 h of exposure. Our results revealed that the canes of V. pentagona and V. amurensis species are a crucial bio-source of an important stilbene with antimicrobial activity and health benefits.

The establishment of Listeria (L.) monocytogenes within food processing environments constitutes a significant public health concern. This versatile bacterium demonstrates an exceptional capacity to endure challenging environmental conditions in the food processing environment, where contamination of food products regularly occurs. The diverse repertoire of stress resistance genes, the potential to colonize biofilms, and the support of a co-existing microbiota have been proposed as root causes for the survival of L. monocytogenes in food processing environments. In this study, 71 sites were sampled after cleaning and disinfection in a European frozen vegetable processing facility, where L. monocytogenes in-house clones persisted for years. L. monocytogenes and L. innocua were detected by a culture-dependent method at 14 sampling sites, primarily on conveyor belts and associated parts. The presence of biofilms, as determined by the quantification of bacterial load and the analysis of extracellular matrix components (carbohydrates, proteins, extracellular DNA) was confirmed at nine sites (12.7%). In two cases, L. innocua was detected in a biofilm. Furthermore, we explored the resident microbial community in the processing environment and on biofilm-positive sites, as well as the co-occurrence of bacterial taxa with Listeria by 16S rRNA gene sequencing. Pseudomonas, Acinetobacter, and Exiguobacterium dominated the microbial community of the processing environment. Using differential abundance analysis, amplicon sequence variants (ASVs) assigned to Enterobacterales (Enterobacter, Serratia, unclassified Enterobacteriaceae) and Carnobacterium were found to be significantly higher abundant in Listeria-positive samples. Several Pseudomonas ASVs were less abundant in Listeria-positive compared to Listeria-negative samples. Acinetobacter, Pseudomonas, Janthinobacterium, Brevundimonas, and Exiguobacterium were key players in the microbial community in biofilms, and Exiguobacterium and Janthinobacterium were more relatively abundant in biofilms. Further, the microbial composition varied between the different areas and the surface materials.