The role of Listeria monocytogenes PstA in ß-lactam resistance requires the cytochrome bd oxidase activity.

c-di-AMP is an essential second messenger that binds and regulates several proteins of different functions within bacterial cells. Among those, PstA is a structurally conserved c-di-AMP-binding protein, but its function is largely unknown. PstA is structurally similar to PII signal transduction proteins, although it specifically binds c-di-AMP rather than other PII ligands such as ATP and α-ketoglutarate. In Listeria monocytogenes, we found that PstA increases ß-lactam susceptibility at normal and low c-di-AMP levels, but increases ß-lactam resistance upon c-di-AMP accumulation. Examining a PstA mutant defective for c-di-AMP binding, we found the apo form of PstA to be toxic for ß-lactam resistance, and the c-di-AMP-bound form to be beneficial. Intriguingly, a role for PstA in ß-lactam resistance is only prominent in aerobic cultures, and largely diminished under hypoxic conditions, suggesting that PstA function is linked to aerobic metabolism. However, PstA does not control aerobic growth rate, and has a modest influence on the tricarboxylic acid cycle and membrane potential-an indicator of cellular respiration. The regulatory role of PstA in ß-lactam resistance is unrelated to reactive oxygen species or oxidative stress. Interestingly, during aerobic growth, PstA function requires the cytochrome bd oxidase (CydAB), a component of the respiratory electron transport chain. The requirement for CydAB might be related to its function in maintaining a membrane potential, or redox stress response activities. Altogether, we propose a model in which apo-PstA diminishes ß-lactam resistance by interacting with an effector protein, and this activity can be countered by c-di-AMP binding or a by-product of redox stress. IMPORTANCE: PstA is a structurally conserved c-di-AMP-binding protein that is broadly present among Firmicutes bacteria. Furthermore, PstA binds c-di-AMP at high affinity and specificity, indicating an important role in the c-di-AMP signaling network. However, the molecular function of PstA remains elusive. Our findings reveal contrasting roles of PstA in ß-lactam resistance depending on c-di-AMP-binding status. We also define physiological conditions for PstA function during aerobic growth. Future efforts can exploit these conditions to identify PstA interaction partners under ß-lactam stress.

Anti-biofilm effect of enzymatic hydrolysates of ovomucin in Listeria monocytogenes and Staphylococcus aureus.

Despite modern advances in food hygiene, food poisoning due to microbial contamination remains a global problem, and poses a great threat to human health. Especially, Listeria monocytogenes and Staphylococcus aureus are gram-positive bacteria found on food-contact surfaces with biofilms. These foodborne pathogens cause a considerable number of food poisoning and infections annually. Ovomucin (OM) is a water-insoluble gel-type glycoprotein in egg whites. Enzymatic hydrolysis can be used to improve the bioactive properties of OM. This study aimed to investigate whether ovomucin hydrolysates (OMHs) produced using five commercial enzymes (Alcalase®, Bromelain, α-Chymotrypsin, Papain, and Pancreatin) can inhibit the biofilm formation of L. monocytogenes ATCC 15313, L. monocytogenes H7962, S. aureus KCCM 11593, and S. aureus 7. Particularly, OMH prepared with papain (OMPP; 500 µg/mL) significantly inhibited biofilm formation in L. monocytogenes ATCC 15313, L. monocytogenes H7962, S. aureus KCCM 11593, and S. aureus 7 by 85.56 %, 80.28 %, 91.70 %, and 79.00 %, respectively. In addition, OMPP reduced the metabolic activity, exopolysaccharide production (EPS), adhesion ability, and gene expression associated with the biofilm formation of these bacterial strains. These results suggest that OMH, especially OMPP, exerts anti-biofilm effects against L. monocytogenes and S. aureus. Therefore, OMPP can be used as a natural anti-biofilm agent to control food poisoning in the food industry.

Attenuation of biofilm and virulence factors of Pseudomonas aeruginosa by tetramethylpyrazine-gold nanoparticles.

Pseudomonas aeruginosa is often identified as the causative agent in nosocomial infections. Their adapted resistance makes them strong towards antimicrobial treatments. They protect and empower their survival behind strong biofilm architecture that works as their armor toward antimicrobial therapy. Additionally, P. aeruginosa generates virulence factors, contributing to chronic infection and recalcitrant phenotypic characteristics. The current study utilizes the benevolence of nanotechnology to develop an alternate technique to control the spreading of P. aeruginosa by limiting its biofilm and virulence development. This study used a natural compound, tetramethylpyrazine, to generate gold nanoparticles. Tetramethylpyrazine-gold nanoparticles (Tet-AuNPs) were presented in spherical shapes, with an average size of 168 ± 52.49 nm and a zeta potential of -12.22 ± 2.06 mV. The minimum inhibition concentration (MIC) of Tet-AuNPs that proved more than 90 % effective in inhibiting P. aeruginosa was 256 µg/mL. Additionally, it also shows antibacterial activities against Staphylococcus aureus (MIC, 256 µg/mL), Streptococcus mutans (MIC, 128 µg/mL), Klebsiella pneumoniae (MIC, 128 µg/mL), Listeria monocytogenes (MIC, 256 µg/mL), and Escherichia coli (MIC, 256 µg/mL). The sub-MIC values of Tet-AuNPs significantly inhibited the early-stage biofilm formation of P. aeruginosa. Moreover, this concentration strongly affected hemolysis, protease activity, and different forms of motilities in P. aeruginosa. Additionally, Tet-AuNPs destroyed the well-established mature biofilm of P. aeruginosa. The expression of genes linked with the biofilm formation and virulence in P. aeruginosa treated with sub-MIC doses of Tet-AuNPs was shown to be significantly suppressed. Gene expression studies support biofilm- and virulence-suppressing effects of Tet-AuNPs at the phenotypic level.

Gentamicin combination treatment is associated with lower mortality in patients with invasive listeriosis: a retrospective analysis.

PURPOSE: Listeria monocytogenes causes severe bacterial infections with the highest mortality rate among foodborne pathogens in Europe. Combination treatment with ampicillin and gentamicin is recommended for invasive manifestations. However, evidence to support this treatment approach remains limited due to a lack of randomised controlled trials. To explore this critical issue further, we conducted this retrospective, single-center study. METHODS: We identified all patients hospitalized with invasive listeriosis at the University Medical Center Hamburg-Eppendorf between 2009 and 2020 and analyzed the effect of gentamicin combination treatment versus monotherapy on 90-day mortality. RESULTS: In total, 36 patients with invasive listeriosis were included, of which 21 patients received gentamicin combination treatment and 15 received monotherapy. The mean age-adjusted Charlson Comorbidity Index (aaCCI) value was lower in the gentamicin combination treatment group (5.4 vs. 7.4). Neurolisteriosis was more common in the gentamicin group (81% vs. 20%). The 90-day mortality was with significantly lower in the gentamicin combination treatment group (10%) compared to the monotherapy group (60%). Multivariable cox regression analysis, adjusted for a propensity score computed based on neurolisteriosis, aaCCI and sex, revealed a significantly reduced hazard ratio of 0.07 (95% CI: 0.01-0.53, p = 0.01) for 90-day mortality for the gentamicin combination treatment. CONCLUSION: This retrospective study highlights the benefit of gentamicin combination treatment in reducing the 90-day mortality rate among patients with invasive listeriosis. The high prevalence of monotherapy in this study cohort raises concerns about the adequacy of antibiotic therapy in clinical practice.

Bacteriocinogenic anti-listerial properties and safety assessment of Enterococcus faecium and Lactococcus garvieae strains isolated from Brazilian artisanal cheesemaking environment.

AIMS: This study aimed to prospect and isolate lactic acid bacteria (LAB) from an artisanal cheese production environment, to assess their safety, and to explore their bacteriocinogenic potential against Listeria monocytogenes. METHODS AND RESULTS: Samples were collected from surfaces of an artisanal-cheese production facility and after rep-PCR and 16S rRNA sequencing analysis, selected strains were identified as to be belonging to Lactococcus garvieae (1 strain) and Enterococcus faecium (14 isolates, grouped into three clusters) associated with different environments (worktables, cheese mold, ripening wooden shelves). All of them presented bacteriocinogenic potential against L. monocytogenes ATCC 7644 and were confirmed as safe (γ-hemolytic, not presenting antibiotic resistance, no mucus degradation properties, and no proteolytic or gelatinase enzyme activity). Additionally, cell growth, acidification and bacteriocins production kinetics, bacteriocin stability in relation to different temperatures, pH, and chemicals were evaluated. According to performed PCR analysis all studied strains generated positive evidence for the presence of entA and entP genes (for production of enterocins A and enterocins P, respectively). However, pediocin PA-1 associated gene was recorded only in DNA obtained from E. faecium ST02JL and Lc. garvieae ST04JL. CONCLUSIONS: It is worth considering the application of these safe LAB or their bacteriocins in situ as an alternative means of controlling L. monocytogenes in cheese production environments, either alone or in combination with other antimicrobials.

Bacteriocins attenuate Listeria monocytogenes-induced intestinal barrier dysfunction and inflammatory response.

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 ß (IL-1ß) 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.

Prevalence, antibiogram and molecular characterization of Listeria monocytogenes from ruminants and humans in New Valley and Beheira Governorates, Egypt.

BACKGROUND: Listeriosis is a global health threat to both animals and humans, especially in developing countries. This study was designed to isolate Listeria monocytogenes from faeces; environmental samples; and cow, sheep and goat milk, as well as human stool, to study its molecular characteristics and antibiotic sensitivity in the New Valley and Beheira Governorates, Egypt. The isolation and identification of L. monocytogenes were carried out using traditional culture and biochemical methods, followed by antibiography, genus confirmation of some isolates and detection and sequencing of InlB genes via PCR. RESULTS: Out of 2097 examined samples, the prevalence of L. monocytogenes was 13.4% in animals; the prevalence was 9.2%, 2.4%, 25.4%, 4%, 42.4%, and 6.4% in cattle faeces, cattle milk, sheep faeces, sheep milk, goat faeces, and goat milk, respectively. However, the prevalence of L. monocytogenes was 8.3% in human samples. Both animal and human isolates showed 100% resistance to trimethoprim-sulfamethoxazole, and the isolates showed the highest sensitivity to flumequine (100%), amikacin (99.2%), gentamicin (97.6%), and levofloxacin (94.6%). Multidrug resistance (MDR) was detected in 86.9% of the tested isolates. The 16 S rRNA and inlB genes were detected in 100% of the randomly selected L. monocytogenes isolates. Phylogenetic analysis of three isolates based on the inlB gene showed 100% identity between faecal, milk and human stool isolates. CONCLUSIONS: Faeces and milk are major sources of listeriosis, and the high degree of genetic similarity between animal and human isolates suggests the possibility of zoonotic circulation. The high prevalence of MDR L. monocytogenes in both animal and human samples could negatively impact the success of prevention and treatments for animal and human diseases, thereby imposing serious risks to public health.

Exploring Citronella's inhibitory mechanism against Listeria monocytogenes and its utilization in preserving cheese.

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.

The synergistic bactericidal effect of simultaneous 222 nm krypton-chlorine excilamp and 307 nm UVB light treatment on sliced cheese and its mechanisms.

In this study, we investigated the combined effect of 222 nm krypton-chlorine excilamp (EX) and 307 nm ultraviolet-B (UVB) light on the inactivation of Salmonella Typhimurium and Listeria monocytogenes on sliced cheese. The data confirmed that simultaneous exposure to EX and UVB irradiation for 80 s reduced S. Typhimurium and L. monocytogenes population by 3.50 and 3.20 log CFU/g, respectively, on sliced cheese. The synergistic cell count reductions in S. Typhimurium and L. monocytogenes in the combined treatment group were 0.88 and 0.59 log units, respectively. The inactivation mechanism underlying the EX and UVB combination treatment was evaluated using fluorescent staining. The combination of EX and UVB light induced the inactivation of reactive oxygen species (ROS) defense enzymes (superoxide dismutase) and synergistic ROS generation, resulting in synergistic lipid peroxidation and destruction of the cell membrane. There were no significant (P > 0.05) differences in the color, texture, or sensory attributes of sliced cheese between the combination treatment and control groups. These results demonstrate that combined treatment with EX and UVB light is a potential alternative strategy for inactivating foodborne pathogens in dairy products without affecting their quality.

Superhydrophobic coatings reduce human bacterial foodborne pathogen attachment to woods used in fresh produce harvest and postharvest packing.

Wood is reportedly more difficult to maintain in hygienic condition versus other food contact materials, yet its use in produce packing and retail warrants efforts to reduce the risk of microbial pathogen contamination and attachment. This study characterized antifouling capabilities of fluorinated silanes applied to wood used in fresh edible produce handling to render the wood superhydrophobic and less supportive of bacterial pathogen attachment. Pine and oak cubic coupon surfaces were treated with 1% (w/w) silane or left untreated. Treated and untreated coupons were inoculated with Salmonella enterica or Listeria monocytogenes and held to facilitate pathogen attachment for 1, 4, or 8 h. Silane treatment of wood produced significant reductions in the proportions of strongly attaching cells for both pathogens versus loosely attaching cells (P < 0.01). Salmonella attachment demonstrated a dependency on wood treatment; silane-treated wood supported a lower fraction of strongly adhering cells (1.87 ± 1.24 log CFU/cm2) versus untreated wood (3.72 ± 0.67 log CFU/cm2). L. monocytogenes demonstrated significant declines in strongly attaching cells during extended exposure to silane-treated wood, from 7.59 ± 0.14 to 5.27 ± 0.68 log CFU/cm2 over 8 h post-inoculation. Microscopic analysis demonstrated silane treatment increased the surface roughness of both woods, leading to superhydrophobic conditions on wood surfaces, consequently decreasing strong attachment of pathogenic bacteria.

Application of gelatin-based zinc oxide nanoparticles bionanocomposite coatings to control Listeria monocytogenes in Talaga cheese and camel meat during refrigerated storage.

Listeria monocytogenes is a concerning foodborne pathogen incriminated in soft cheese and meat-related outbreaks, highlighting the significance of applying alternative techniques to control its growth in food. In the current study, eco-friendly zinc oxide nanoparticles (ZnO-NPs) were synthesized using Rosmarinus officinalis, Punica granatum, and Origanum marjoram extracts individually. The antimicrobial efficacy of the prepared ZnO-NPs against L. monocytogenes was assessed using the agar well diffusion technique. Data indicated that ZnO-NPs prepared using Origanum marjoram were the most effective; therefore, they were used for the preparation of gelatin-based bionanocomposite coatings. Furthermore, the antimicrobial efficacy of the prepared gelatin-based bionanocomposite coatings containing eco-friendly ZnO-NPs was evaluated against L. monocytogenes in Talaga cheese (an Egyptian soft cheese) and camel meat during refrigerated storage at 4 ± 1 oC. Talaga cheese and camel meat were inoculated with L. monocytogenes, then coated with gelatin (G), gelatin with ZnO-NPs 1% (G/ZnO-NPs 1%), and gelatin with ZnO-NPs 2% (G/ZnO-NPs 2%). Microbiological examination showed that the G/ZnO-NPs 2% coating reduced L. monocytogenes count in the coated Talaga cheese and camel meat by 2.76 ± 0.19 and 2.36 ± 0.51 log CFU/g, respectively, by the end of the storage period. Moreover, G/ZnO-NPs coatings controlled pH changes, reduced water losses, and improved the sensory characteristics of Talaga cheese and camel meat, thereby extending their shelf life. The obtained results from this study indicate that the application of gelatin/ZnO-NPs 2% bionanocomposite coating could be used in the food industry to control L. monocytogenes growth, improve quality, and extend the shelf life of Talaga cheese and camel meat.

Enhancing functional buffalo yogurt: Improving physicochemical properties, biological activities, and shelf life using marjoram and geranium essential oils.

The use of essential oils (EO) has attracted interest in the food industry because of their wide range of beneficial properties. In this study, a new functional yogurt was developed using 2 EO, marjoram and geranium, at 3 different concentrations (0.2%, 0.4%, and 0.6% vol/vol). The physicochemical properties, including syneresis, viscosity, pH, and chemical composition; bioactivities, including antioxidant activity, anticancer and antibacterial effects, total phenolic content (TPC), and total flavonoid content (TFC); and sensory characteristics of the developed yogurt were evaluated. The findings indicated that the yogurts fortified with 0.6% marjoram or geranium exhibited higher viscosity and lower syneresis compared with other treatments. The yogurt supplemented with 0.6% marjoram displayed significant antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli. In addition, the yogurt enriched with geranium and marjoram oils at a concentration of 0.6% had notably significant (P < 0.05) higher TFC levels compared with the control sample and other concentrations. In the same context, in terms of TPC, yogurt supplemented with 0.6% marjoram displayed significantly (P < 0.05) elevated levels in comparison to the other samples tested. Yogurt enriched with marjoram oil exhibited noteworthy antioxidant activity, followed by geranium oil, compared with the control samples. The yogurt supplemented with 0.6% marjoram demonstrated strong radical scavenging activity, and the yogurt fortified with 0.6% geranium showed higher anticancer activity against HepG2 human liver carcinoma cells and oxidative stress enzyme activities. Among the various concentrations of EO tested, the yogurts fortified with 0.6% marjoram or geranium EO exhibited the most favorable outcomes, followed by 0.4% marjoram or geranium. To summarize, geranium and marjoram EO can be used as a potential nutritious ingredient and as a natural preservative for milk and related products.

Characterization of Antimicrobial Activities of Bifidobacterium lactis BB-12 and Their Inhibitory Effect Against Some Foodborne Pathogens.

Bifidobacterium animalis subsp. lactis BB-12, a probiotic, has shown potential to promote health benefits and control pathogens. This study aimed to investigate the effectiveness of BB-12 and its cell-free supernatant (CFS) in inhibiting the growth of Listeria monocytogenes and Salmonella enterica serovar Typhimurium. To assess the antimicrobial activity of BB-12, agar well diffusion, disk diffusion, and minimum inhibitory concentration (MIC) tests were conducted. The bicinchoninic acid (BCA) assay was performed to measure the protein concentration in CFS. The study's results indicated that the BB-12 strain inhibited the pathogens' growth. The disk diffusion test using BB-12 showed inhibitory results ranging from 11 to 14 mm for both bacteria. The agar well diffusion test reported the zone of inhibition ranging from 11.6 to 16 mm for both bacteria. The MIC test was conducted as a confirmatory test, which demonstrated the highest inhibitory zone using 2 McFarland (6 × 108 CFU/mL) concentrations of probiotics on L. monocytogenes (44.98%) and S. Typhimurium (66.41%). The disk diffusion test revealed that the probiotic CFS had a significant inhibitory impact on S. Typhimurium with a 16.6 mm zone of inhibition. The BCA test findings indicated that the 24- and 48-h CFSs exhibited inhibitory properties against infections. Notably, the 24-h CFS, including a protein level of 78.47 µg/mL, demonstrated a more pronounced inhibitory impact on both pathogens. The findings highlight that utilizing the BB-12 strain and its CFS can serve as a viable approach to battle infections, enhancing food safety and public health.

Laser-induced 2D/0D graphene-nanoceria freestanding paper-based films for on-site hydrogen peroxide monitoring in no-touch disinfection treatments.

A one-shot CO2 laser-based strategy to generate conductive reduced graphene oxide (rGO) decorated with nanoceria (nCe) is proposed. The 2D/0D rGO-nCe films, integrated as catalytic sensing layers in paper-based sensors, were employed for on-site monitoring of indoor fogging treatments against Listeria monocytogenes (Lm), a ubiquitous pathogenic bacterium. The rGO-nCe laser-assisted synthesis was optimized to preserve the rGO film morphological and electron-transfer features and simultaneously integrate catalytic nCe. The films were characterized by microscopical (SEM), spectroscopical (EDX, Raman, and FTIR), and electrochemical techniques. The most performing film was integrated into a nitrocellulose substrate, and the complete sensor was assembled via a combination of xurography and stencil printing. The rGO-nCe sensor's catalytic activity was proved toward the detection of H2O2, obtaining sensitive determination (LOD = 0.3 µM) and an extended linear range (0.5-1500 µM). Eventually, the rGO-nCe sensor was challenged for the real-time continuous monitoring of hydrogen peroxide aerosol during no-touch fogging treatment conducted following the EU's recommendation for biocidal product use. Treatment effectiveness was proved toward three Lm strains characterized by different origins, i.e., type strain ATCC 7644, clinical strain 338, and food strain 641/6II. The sensor allows for discrimination and quantification treatments at different environmental biocidal amounts and fogging times, and correlates with the microbiological inhibition, promoting the proposed sensor as a useful tool to modulate and monitor no-touch treatments.

Antibacterial and Antioxidant Activity of Synthetic Polyoxygenated Flavonoids.

Flavonoids are an abundant class of naturally occurring compounds with broad biological activities, but their limited abundance in nature restricts their use in medicines and food additives. Here we present the synthesis and determination of the antibacterial and antioxidant activities of twenty-two structurally related flavonoids (five of which are new) by scientifically validated methods. Flavanones (FV1-FV11) had low inhibitory activity against the bacterial growth of MRSA 97-7. However, FV2 (C5,7,3',4' = OH) and FV6 (C5,7 = OH; C4' = SCH3) had excellent bacterial growth inhibitory activity against Gram-negative E. coli (MIC = 25 µg/mL for both), while Chloramphenicol (MIC = 25 µg/mL) and FV1 (C5,7,3' = OCH3; 4' = OH) showed inhibitory activity against Gram-positive L. monocytogenes (MIC = 25 µg/mL). From the flavone series (FO1-FO11), FO2 (C5,7,3',4' = OH), FO3 (C5,7,4' = OH; 3' = OCH3), and FO5 (C5,7,4' = OH) showed good inhibitory activity against Gram-positive MRSA 97-7 (MIC = 50, 12, and 50 µg/mL, respectively), with FO3 being more active than the positive control Vancomycin (MIC = 25 µg/mL). FO10 (C5,7= OH; 4' = OCH3) showed high inhibitory activity against E. coli and L. monocytogenes (MIC = 25 and 15 µg/mL, respectively). These data add significantly to our knowledge of the structural requirements to combat these human pathogens. The positions and number of hydroxyl groups were key to the antibacterial and antioxidant activities.

Wild Vitis Species as Stilbenes Sources: Cane Extracts and Their Antibacterial Activity against Listeria monocytogenes.

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.

In vitro antimicrobial, antibiofilm photodynamic activity, and molecular dynamic simulations of tetra-cationic porphyrinmembrane interactions against foodborne microorganisms.

This manuscript presents a new report on the in vitro antimicrobial photo-inactivation of foodborne microorganisms (Salmonella spp. and Listeria monocytogenes) using tetra-cationic porphyrins. Isomeric tetra-cationic porphyrins (3MeTPyP, 4MeTPyP, 3PtTPyP, and 4PtTPyP) were tested, and antimicrobial activity assays were performed at specific photosensitizer concentrations under dark and white-light LED irradiation conditions. Among the tested bacterial strains, 4MeTPyP exhibited the highest efficiency, inhibiting bacterial growth within just 60 min at low concentrations (17.5 µM). The minimal inhibitory concentration of 4MeTPyP increased when reactive oxygen species scavengers were present, indicating the significant involvement of singlet oxygen species in the photooxidation mechanism. Furthermore, the checkerboard assay testing the association of 4MeTPyP showed an indifferent effect. Atomic force microscopy analyses and dynamic simulations were conducted to enhance our understanding of the interaction between this porphyrin and the strain's membrane.

Improving the Composition and Bioactivity of Cocoa (Theobroma cacao L.) Bean Shell Extract by Choline Chloride-Lactic Acid Natural Deep Eutectic Solvent Extraction Assisted by Pulsed Electric Field Pre-Treatment.

An environmentally friendly method for the release of cocoa bean shell (CBS) extracts is proposed in this paper. This work aims to investigate the effect of pulsed electric field (PEF) pre-treatment on subsequent solid-liquid extraction (SLE) of metabolites with choline chloride-lactic acid natural deep eutectic solvent (NaDES) and bioactivity of cocoa bean shell (CBS) extract. Two different media for PEF application were evaluated: water and chlorine chloride-lactic acid. Total polyphenols (TPC), total flavonoids (TFC), individual major compounds, and antioxidant and antibacterial activity of CBS extracts were assessed. The performance of PEF-assisted extraction was compared with SLE and ultrasound-assisted extraction (UAE). The proposed method improved the release of TPC up to 45% and TFC up to 48% compared with the conventional extraction. The CBS extract showed medium growth inhibition of Escherichia coli and high growth inhibition of Salmonella sp, Listeria monocytogenes, and Staphylococcus aureus. Thus, an extract with enhanced antioxidant and antibacterial properties was obtained.

Changes in the ability of Listeria monocytogenes to resist thermal treatment and simulated gastric condition after exposure to sequential stresses in minced meat.

In this study, Listeria monocytogenes from minced pork was evaluated for changes in resistance to thermal treatment and gastric fluid following environmental stresses during food processing. Bacteria were exposed to cold stress, followed by successive exposures to different stressors (lactic acid (LA), NaCl, or Nisin), followed by thermal treatments, and finally, their gastrointestinal tolerance was determined. Adaptation to NaCl stress reduced the tolerance of L. monocytogenes to subsequent LA and Nisin stress. Adaptation to LA stress increased bacterial survival in NaCl and Nisin-stressed environments. Bacteria adapted to Nisin stress showed no change in tolerance to subsequent stress conditions. In addition, treatment with NaCl and LA enhanced the thermal tolerance of L. monocytogenes, but treatment with Nisin decreased the thermal tolerance of the bacteria. Almost all of the sequential stresses reduced the effect of a single stress on bacterial thermal tolerance. The addition of LA and Nisin as a second step of stress reduced the tolerance of L. monocytogenes to gastric fluid, whereas the addition of NaCl enhanced its tolerance. The results of this study are expected to inform processing conditions and sequences for meat preservation and processing and reduce uncertainty in risk assessment of foodborne pathogens due to stress adaptation.

Chemical mutagenesis of Listeria monocytogenes for increased tolerance to benzalkonium chloride shows independent genetic underpinnings and off-target antibiotic resistance.

Listeria monocytogenes, a potentially fatal foodborne pathogen commonly found in food processing facilities, creates a significant economic burden that totals more than $2 billion annually in the United States due to outbreaks. Quaternary ammonium compounds (QACs), including benzalkonium chloride (BAC), are among the most widely used sanitizers to inhibit the growth and spread of L. monocytogenes from food processing facilities. However, resistance to QACs has been increasing in L. monocytogenes and different genetic mechanisms conferring resistance have been discovered. Here, we used ethyl methanesulfonate (EMS) to chemically mutagenize the BAC-susceptible strain, L. monocytogenes FSL-N1-304. We isolated two mutants with increased tolerance to BAC compared to the parental strain. Next, we assessed the off-target effect of increased tolerance to BAC by measuring the minimum inhibitory concentrations (MICs) of a diverse set of antibiotics, revealing that mut-1 and mut-2 displayed significantly increased resistance to fluoroquinolone antibiotics compared to the parental strain. A hemolysis assay was then used to investigate a potential correlation between BAC tolerance and virulence. Interestingly, mut-1 and mut-2 both exhibited significantly higher hemolysis percentage than the parental strain. We then sequenced the genomes of the parental strain and both mutants to identify mutations that may be involved in the increased resistance to BAC. We identified 3 and 29 mutations in mut-1 and mut-2, respectively. mut-1 contained nonsynonymous mutations in dagK (a diacylglycerol kinase), lmo2768 (a permease-encoding gene), and lmo0186 (resuscitation promoting factor). mut-2 contained a nonsense mutation in the nucleotide excision repair enzyme UvrABC system protein B encoding gene, uvrB, which likely accounts for the higher number of mutations observed. Transcriptome analysis in the presence of BAC revealed that genes related to the phosphotransferase system and internalins were up-regulated in both mutants, suggesting their significance in the BAC stress response. These two mutants provide insights into alternative mechanisms for increased BAC tolerance and could further our understanding of how L. monocytogenes persists in the food processing environment.