As a common foodborne pathogen, infection with L. monocytogenes poses a significant threat to human life and health. The objective of this study was to employ comparative genomics to unveil the biodiversity and evolutionary characteristics of L. monocytogenes strains from different regions, screening for potential target genes and mining novel target genes, thus providing significant reference value for the specific molecular detection and therapeutic targets of L. monocytogenes strains. Pan-genomic analysis revealed that L. monocytogenes from different regions have open genomes, providing a solid genetic basis for adaptation to different environments. These strains contain numerous virulence genes that contribute to their high pathogenicity. They also exhibit relatively high resistance to phosphonic acid, glycopeptide, lincosamide, and peptide antibiotics. The results of mobile genetic elements indicate that, despite being located in different geographical locations, there is a certain degree of similarity in bacterial genome evolution and adaptation to specific environmental pressures. The potential target genes identified through pan-genomics are primarily associated with the fundamental life activities and infection invasion of L. monocytogenes, including known targets such as inlB, which can be utilized for molecular detection and therapeutic purposes. After screening a large number of potential target genes, we further screened them using hub gene selection methods to mining novel target genes. The present study employed eight different hub gene screening methods, ultimately identifying ten highly connected hub genes (bglF_1, davD, menE_1, tilS, dapX, iolC, gshAB, cysG, trpA, and hisC), which play crucial roles in the pathogenesis of L. monocytogenes. The results of pan-genomic analysis showed that L. monocytogenes from different regions exhibit high similarity in bacterial genome evolution. The PCR results demonstrated the excellent specificity of the bglF_1 and davD genes for L. monocytogenes. Therefore, the bglF_1 and davD genes hold promise as specific molecular detection and therapeutic targets for L. monocytogenes strains from different regions.

The engineering of a home-made portable double-layer filtration and concentration device with the common syringe for rapid analysis of water samples is reported. The core elements of the device were two installed filtration membranes with different pore sizes for respective functions. The upper filtration membrane was used for preliminary intercepting large interfering impurities (interception membrane), while the lower filtration membrane was used for collecting multiple target pathogens (enrichment membrane) for determination. This combination can make the contaminated environmental water, exemplified by surface water, filtrated quickly through the device and just retained the target bacteria of Escherichia coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes on the lower enrichment membrane. Integrating with surface-enhanced Raman spectra (SERS) platform to decode the SERS-Tags (SERS-TagCVa, SERS-TagR6G, and SERS-TagMB) already labeled on each of the enriched bacteria based the antibody-mediated immuno-recognition effect, fast separation, concentration, and detection of multiple pathogenic bacteria from the bulk of contaminated environmental water were realized. Results show that within 30 min, all target bacteria in the lake water can be simultaneously and accurately measured in the range from 101 to 106 CFU mL-1 with detection limit of 10.0 CFU mL-1 without any pre-culture procedures. This work highlights the simplicity, rapidness, cheapness, selectivity, and the robustness of the constructed method for simultaneous detecting multiple pathogens in aqueous samples. This protocol opens a new avenue for facilitating the development of versatile analytical tools for drinking water and food safety monitoring in underdeveloped or developing countries.

Pre-cut fresh fruits and vegetables are highly appealing to consumers for their convenience, however, as they are highly susceptible to microbial contamination in processing, the potential risks of foodborne illnesses to public health are not negligible. This study aimed to assess the prevalence, antibiotic susceptibility and molecular characteristics of major foodborne pathogens (Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Salmonella) isolated from fresh-cut fruits and vegetables in Beijing, China. 86 stains were isolated from 326 samples, with S. aureus being the highest prevalence (15.38 %), followed by E. coli (9.23 %) and L. monocytogenes (1.85 %), while no Salmonella was detected. The prevalence by type of food indicated that fruit trays and mixed vegetables were more susceptible to contamination by pathogens. 98 % of S. aureus were resistant to at least of one antibiotic, and showed a high resistance rate to benzylpenicillin (90 %) and oxacillin (48 %). Among 25 E. coli isolates, 57.67 % of which exhibited multi-drug resistance, with common resist to trimethoprim/sulfamethoxazole (66.67 %) and ampicillin (63.33 %). A total of 9 sequence types (STs) and 8 spa types were identified in 35 S. aureus isolates, with ST398-t34 being the predominant type (42.86 %). Additionally, analysis of 25 E. coli isolates demonstrated significant heterogeneity, characterized by 22 serotypes and 18 STs. Genomic analysis revealed that 5 and 44 distinct antibiotic resistance genes (ARGs) in S. aureus and E. coli, respectively. Seven quinolone resistance-determining regions (QRDRs) mutations were identified in E. coli isolates, of which GyrA (S83L) was the most frequently detected. All the S. aureus and E. coli isolates harbored virulence genes. ARGs in S. aureus and E. coli showed a significant positive correlation with plasmids. Furthermore, one L. monocytogenes isolate, which was ST101 and serogroupIIc from watermelon sample, harbored virulence genes (inlA and inlB) and LIPI-1 pathogenic islands (prfA, plcA, hly and actA), which posed potential risks for consumer\'s health. This study focused on the potential microbial risk of fresh-cut fruits and vegetables associated with foodborne diseases, improving the scientific understanding towards risk assessment related to ready-to-eat foods.

Bacteriocins have the potential to effectively improve food-borne infections or gastrointestinal diseases and hold promise as viable alternatives to antibiotics. This study aimed to explore the antibacterial activity of three bacteriocins (nisin, enterocin Gr17, and plantaricin RX-8) and their ability to attenuate intestinal barrier dysfunction and inflammatory responses induced by Listeria monocytogenes, respectively. Bacteriocins have shown excellent antibacterial activity against L. monocytogenes without causing any cytotoxicity. Bacteriocins inhibited the adhesion and invasion of L. monocytogenes on Caco-2 cells, lactate dehydrogenase (LDH), trans-epithelial electrical resistance (TEER), and cell migration showed that bacteriocin improved the permeability of Caco-2 cells. These results were attributed to the promotion of tight junction proteins (TJP) assembly, specifically zonula occludens-1 (ZO-1), occludin, and claudin-1. Furthermore, bacteriocins could alleviate inflammation by inhibiting the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways and reducing the secretion of interleukin-6 (IL-6), interleukin-1 β (IL-1β) and tumor necrosis factor α (TNF-α). Among three bacteriocins, plantaricin RX-8 showed the best antibacterial activity against L. monocytogenes and the most pronounced protective effect on the intestinal barrier due to its unique structure. Based on our findings, we hypothesized that bacteriocins may inhibit the adhesion and invasion of L. monocytogenes by competing adhesion sites. Moreover, they may further enhance intestinal barrier function by inhibiting the expression of L. monocytogenes virulence factors, increasing the expression of TJP and decreasing the secretion of inflammatory factors. Therefore, bacteriocins will hopefully be an effective alternative to antibiotics, and this study provides valuable insights into food safety concerns. KEY POINTS: • Bacteriocins show excellent antibacterial activity against L. monocytogenes • Bacteriocins improve intestinal barrier damage and inflammatory response • Plantaricin RX-8 has the best protective effect on Caco-2 cells damage.

Nowadays, the discovery of alternative natural antimicrobial substances such as bacteriophages, essential oils, and other physical and chemical agents is developing in the food industry. In this study, nine bacteriophages were isolated from various parts of raw chickens and exhibited lytic activities against L. monocytogenes and various Listeria spp. The characterization of phage vB_LmoS-PLM9 was stable at 4 to 50 °C and pH range from 4 to 10. Phage vB_LmoS-PLM9 had a circular, double-stranded genomic DNA with 38,345 bp having endolysin but no antibiotic resistance or virulence genes. Among the eight essential oils tested at 10 %, cinnamon bark, and cassia oils showed the strongest antilisterial activities. The combined use of phage vB_LmoS-PLM9 and cinnamon oils indicated higher efficiency than single treatments. The combination of phage (MOI of 10) and both cinnamon oils (0.03 %) reduced the viable counts of L. monocytogenes and inhibited the regrowth of resistant cell populations in broth at 30 °C. Furthermore, treatment with the combination of phage (MOI of 100) and cinnamon oil (0.125 %) was effective in milk, especially at 4 °C by reducing the viable count to less than lower limit of detection. These results suggest combining phage and cinnamon oil is a potential approach for controlling L. monocytogenes in milk.

Listeria monocytogenes presents significant risk to human health due to its high resistance and capacity to form toxin-producing biofilms that contaminate food. The objective of this study was to assess the inhibitory effect of citronella aldehyde (CIT) on L. monocytogenes and investigate the underlying mechanism of inhibition. The results indicated that the minimum inhibitory concentration (MIC) and Minimum sterilisation concentration (MBC) of CIT against L. monocytogenes was 2 μL/mL. At this concentration, CIT was able to effectively suppress biofilm formation and reduce metabolic activity. Crystalline violet staining and MTT reaction demonstrated that CIT was able to inhibit biofilm formation and reduce bacterial cell activity. Furthermore, the motility assessment assay revealed that CIT inhibited bacterial swarming and swimming. Scanning electron microscopy (SEM) and laser confocal microscopy (LSCM) observations revealed that CIT had a significant detrimental effect on L. monocytogenes cell structure and biofilm integrity. LSCM also observed that nucleic acids of L. monocytogenes were damaged in the CIT-treated group, along with an increase in bacterial extracellular nucleic acid leakage. The proteomic results also confirmed the ability of CIT to affect the expression of proteins related to processes including metabolism, DNA replication and repair, transcription and biofilm formation in L. monocytogenes. Consistent with the proteomics results are ATPase activity and ATP content of L. monocytogenes were significantly reduced following treatment with various concentrations of CIT. Notably, CIT showed good inhibitory activity against L. monocytogenes on cheese via fumigation at 4 °C.This study establishes a foundation for the potential application of CIT in food safety control.

Owing to their lack of outer skin, Chinese bayberries are highly susceptible to mechanical damage during picking, which accelerates bacterial invasion and rotting, shortening their shelf life. In this study, montmorillonite (MMT) was used to absorb an aqueous sodium chlorite solution embedded in a carboxymethyl cellulose sodium hydrogel after freeze drying, and the hydrogel was crosslinked by Al3+ ions. Al3+ hydrolyzed to produce H+, creating an acidic environment within the hydrogel and reacting with NaClO2 to slowly release ClO2. We prepared a ClO2 slow-release hydrogel gasket with 0.5 wt% MMT-NaClO2 and investigated its storage effect on postharvest Chinese bayberries. Its inhibition rates against Escherichia coli and Listeria monocytogenes were 98.84% and 98.96%, respectively. The results showed that the gasket preserved the appearance and nutritional properties of the berries. The antibacterial hydrogel reduced hardness loss by 26.57% and ascorbic acid loss by 46.36%. This new storage method could also be applicable to other fruits and vegetables.

BACKGROUND: Listeria monocytogenes, a Gram-positive bacterium, is a prominent foodborne pathogen that causes listeriosis and poses substantial health hazards worldwide. The continuing risk of listeriosis outbreaks underlies the importance of designing an effective prevention strategy and developing a robust immune response by reverse vaccinology approaches. This study aimed to provide a critical approach for developing a potent multiepitope vaccine against this foodborne disease.
METHODS: A chimeric peptide construct containing 5 B-cell epitopes, 16 major histocompatibility complex I (MHC-I) epitopes, and 18 MHC-II epitopes were used to create a subunit vaccination against L. monocytogenes. The vaccine safety was evaluated by several online methods, and molecular docking was performed using ClusPro to determine the binding affinity. Immune simulation was performed using the C-ImmSimm server to demonstrate the immune response.
RESULTS: The results validated the antigenicity, non-allergenicity, and nontoxicity of the chimeric peptide construct, confirming its suitability as a subunit vaccine. Molecular docking showed a good score of 1276.5 and molecular dynamics simulations confirmed the construct\'s efficacy, demonstrating its promise as a good candidate for listeriosis prophylaxis. The population coverage was as high as 91.04% with a good immune response, indicating good antigen presentation with dendritic cells and production of memory cells.
CONCLUSIONS: The findings of this study highlight the potential of the designed chimeric peptide construct as an effective subunit vaccine against Listeria, paving the way for future advances in preventive methods and vaccine design.

Listeria monocytogenes are considered to be the major foodborne pathogen worldwide. To understand the prevalence and potential risk of L. monocytogenes in retail foods, a total of 1243 retail foods in 12 food categories were sampled and screened for L. monocytogenes from 2020 to 2022 in Huzhou, China. A total of 46 out of 1234 samples were confirmed to be L. monocytogenes positive with a total rate of 3.7%. The contamination rate of seasoned raw meat (15.2%) was the highest, followed by raw poultry meat and raw livestock meat (9.9%) and salmon sashimi (9.5%). The L. monocytogenes isolates belonged to four serotypes, 1/2a,1/2b, 1/2c, and 4b, with the most prevalent serotype being 1/2a (47.9%). All isolates were grouped into 15 sequence types (STs) belonging to 14 clonal complexes (CCs) via multilocus sequence typing (MLST). The most prevalent ST was ST9/CC9 (23.9%), followed by ST3/CC3 (19.6%) and ST121/CC121 (17.4%). Notably, 11 STs were detected from ready-to-eat (RTE) foods, some of them have been verified to be strongly associated with clinical origin listeriosis cases, such as ST3, ST2, ST5, ST8, and ST87. Listeria pathogenicity islands 1 (LIPI-1) and LIPI-2 were detected in approximately all L. monocytogenes isolates, whereas the distribution of both LIPI-3 genes and LIPI-4 genes exhibited association with specific ST, with LIPI-3 in ST3 and ST288, and LIPI-4 in ST87. The strains carrying LIPI-3 and LIPI-4 virulence genes in this study were all isolated from RTE foods. Antimicrobial susceptibility tests showed that >90% of isolates were susceptible to PEN, AMP, ERY, CIP, SXT, VAN, CHL, and GEN, indicating the antibiotic treatment might be still efficient for most of the L. monocytogenes strains. However, for the three clinical first-line antibiotics (PEN, AMP, and GEN), we also observed three and four strains showing MIC values greater than the susceptibility standards for PEN and AMP, respectively, and one strain showing resistance to GEN.

Listeria monocytogenes exhibits varying levels of pathogenicity when entering the host through contaminated food. However, little is known regarding the stress response and environmental tolerance mechanism of different virulence strains to host gastrointestinal (GI) stimuli. This study analyzed the differences in the survival and genes of stress responses among two strains of L. monocytogenes 10403S (serotype 1/2a, highly virulent strain) and M7 (serotype 4a, low-virulence strain) during simulated gastrointestinal digestion. The results indicated that L. monocytogenes 10403S showed greater acid and bile salt tolerance than L. monocytogenes M7, with higher survival rates and less cell deformation and cell membrane permeability during the in vitro digestion. KEGG analysis of the transcriptomes indicated that L. monocytogenes 10403S displayed significant activity in amino acid metabolism, such as glutamate and arginine, associated with acid tolerance. Additionally, L. monocytogenes 10403S demonstrated a higher efficacy in promoting activities that preserve bacterial cell membrane integrity and facilitate flagellar protein synthesis. These findings will contribute valuable practical insights into the tolerance distinctions among different virulence strains of L. monocytogenes in the GI environment.