Signals behind Listeria monocytogenes virulence mechanisms.

The tight and coordinated regulation of virulence gene expression is crucial to ensure the survival and persistence of bacterial pathogens in different contexts within their hosts. Considering this, bacteria do not express virulence factors homogenously in time and space, either due to their associated fitness cost or to their detrimental effect at specific infection stages. To efficiently infect and persist into their hosts, bacteria have thus to monitor environmental cues or chemical cell-to-cell signaling mechanisms that allow their transition from the external environment to the host, and therefore adjust gene expression levels, intrinsic biological activities, and appropriate behaviors. Listeria monocytogenes (Lm), a major Gram-positive facultative intracellular pathogen, stands out for its adaptability and capacity to thrive in a wide range of environments. Because of that, Lm presents itself as a significant concern in food safety and public health, that can lead to potentially life-threatening infections in humans. A deeper understanding of the intricate bacterial virulence mechanisms and the signals that control them provide valuable insights into the dynamic interplay between Lm and the host. Therefore, this review addresses the role of some crucial signals behind Lm pathogenic virulence mechanisms and explores how the ability to assimilate and interpret these signals is fundamental for pathogenesis, identifying potential targets for innovative antimicrobial strategies.

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.

Idiosyncratic invasion trajectories of human bacterial pathogens facing temperature disturbances in soil microbial communities.

Current knowledge about effects of disturbance on the fate of invaders in complex microbial ecosystems is still in its infancy. In order to investigate this issue, we compared the fate of Klebsiella pneumoniae (Kp) and Listeria monocytogenes (Lm) in soil microcosms. We then used environmental disturbances (freeze-thaw or heat cycles) to compare the fate of both invaders and manipulate soil microbial diversity. Population dynamics of the two pathogens was assessed over 50 days of invasion while microbial diversity was measured at times 0, 20 and 40 days. The outcome of invasion was strain-dependent and the response of the two invaders to disturbance differed. Resistance to Kp invasion was higher under the conditions where resident microbial diversity was the highest while a significant drop of diversity was linked to a higher persistence. In contrast, Lm faced stronger resistance to invasion in heat-treated microcosms where diversity was the lowest. Our results show that diversity is not a universal proxy of resistance to microbial invasion, indicating the need to properly assess other intrinsic properties of the invader, such as its metabolic repertoire, or the array of interactions between the invader and resident communities.

Acute response to pathogens in the early human placenta at single-cell resolution.

The placenta is a selective maternal-fetal barrier that provides nourishment and protection from infections. However, certain pathogens can attach to and even cross the placenta, causing pregnancy complications with potential lifelong impacts on the child's health. Here, we profiled at the single-cell level the placental responses to three pathogens associated with intrauterine complications-Plasmodium falciparum, Listeria monocytogenes, and Toxoplasma gondii. We found that upon exposure to the pathogens, all placental lineages trigger inflammatory responses that may compromise placental function. Additionally, we characterized the responses of fetal macrophages known as Hofbauer cells (HBCs) to each pathogen and propose that they are the probable niche for T. gondii. Finally, we revealed how P. falciparum adapts to the placental microenvironment by modulating protein export into the host erythrocyte and nutrient uptake pathways. Altogether, we have defined the cellular networks and signaling pathways mediating acute placental inflammatory responses that could contribute to pregnancy complications.

Comprehensive strategies for controlling Listeria monocytogenes biofilms on food-contact surfaces.

Listeria monocytogenes biofilms formed on food-contact surfaces within food-processing facilities pose a significant challenge, serving as persistent sources of cross-contamination. In this review, we examined documented cases of foodborne outbreaks and recalls linked to L. monocytogenes contamination on equipment surfaces and in the food production environment, provided an overview of the prevalence and persistence of L. monocytogenes in different food-processing facilities, and discussed environmental factors influencing its biofilm formation. We further delved into antimicrobial interventions, such as chemical sanitizers, thermal treatments, biological control, physical treatment, and other approaches for controlling L. monocytogenes biofilms on food-contact surfaces. This review provides valuable insights into the persistent challenge of L. monocytogenes biofilms in food processing, offering a foundation for future research and practical strategies to enhance food safety.

A metallothionein from disk abalone (Haliotis discus discus): Insights into its functional roles in immune response, metal tolerance, and oxidative stress.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential biological markers for assessing metal pollution in various biomonitoring programs. However, the functional properties of these proteins are yet to be comprehensively characterized in most marine invertebrates. In this study, we identified and characterized an MT homolog from the disk abalone (Haliotis discus discus), referred to as disk abalone MT (AbMT). AbMT exhibited the same primary structural features as MTs from other mollusks containing two ß-domains (ß2ß1-form). AbMT protein demonstrated metal-binding and detoxification abilities against Zn, Cu, and Cd, as evidenced by Escherichia coli growth kinetics, metal tolerance analysis, and UV absorption spectrum. Transcriptional analysis revealed that AbMT was ubiquitously expressed in all analyzed tissues and upregulated in gill tissue following challenge with Vibrio parahaemolyticus, Listeria monocytogenes, and viral hemorrhagic septicemia virus (VHSV). Additionally, overexpression of AbMT suppressed LPS-induced NO production in RAW264.7 macrophages, protected cells against H2O2-induced oxidative stress, and promoted macrophage polarization toward the M1 phase. Conclusively, these findings suggest an important role for AbMT in environmental stress protection and immune regulation in disk abalone.

Is the persistence of Listeria monocytogenes in food processing facilities and its resistance to pathogen intervention linked to its phylogeny?

The foodborne pathogen Listeria monocytogenes is differentiated into four distinct lineages which differ in their virulence. It remains unknown, however, whether the four lineages also differ with respect to their ability to persist in food processing facilities, their resistance to high pressure, a preservation method that is used commercially for Listeria control on ready-to-eat meats, and their ability to form biofilms. This study aimed to determine differences in the pressure resistance and biofilm formation of 59 isolates of L. monocytogenes representing lineages I and II. Furthermore, the genetic similarity of 9 isolates of L. monocytogenes that were obtained from a meat processing facility over a period of 1 year and of 20 isolates of L. monocytogenes from food processing facilities was analyzed to assess whether the ability of the lineages of L. monocytogenes to persist in these facilities differs. Analysis of 386 genomes with respect to the source of isolation revealed that genomes of lineage II are over-represented in meat isolates when compared with clinical isolates. Of the 38 strains of Lm. monocytogenes that persisted in food processing facilities (this study or published studies), 31 were assigned to lineage II. Isolates of lineage I were more resistant to treatments at 400 to 600 MPa. The thickness of biofilms did not differ between lineages. In conclusion, strains of lineage II are more likely to persist in food processing facilities while strains of lineage I are more resistant to high pressure.IMPORTANCEListeria monocytogenes substantially contributes to the mortality of foodborne disease in developed countries. The virulence of strains of four lineages of L. monocytogenes differs, indicating that risks associated with the presence of L. monocytogenes are lineage specific. Our study extends the current knowledge by documentation that the lineage-level phylogeny of L. monocytogenes plays a role in the source of isolation, in the persistence in food processing facilities, and in the resistance to pathogen intervention technologies. In short, the control of risks associated with the presence of L. monocytogenes in food is also lineage specific. Understanding the route of contamination L. monocytogenes is an important factor to consider when designing improved control measures.

The trophoblast surface becomes refractory to adhesion by congenitally transmitted Toxoplasma gondii and Listeria monocytogenes during cytotrophoblast to syncytiotrophoblast development.

The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance of understanding these mechanisms and challenges in replicating trophoblast-pathogen interactions using in vitro models, we tested an existing stem-cell-derived model of trophoblast development for its relevance to infection with Toxoplasma gondii. We grew human trophoblast stem cells (TSCT) under conditions leading to either syncytiotrophoblast (TSSYN) or cytotrophoblast (TSCYT) and infected them with T. gondii. We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TSSYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by transmission electron microscopy and scanning electron microscopy (SEM), a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TSSYNs were highly refractory to parasite adhesion and replication, while TSCYTs were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TSSC-derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes. We demonstrate that TSSYNs are highly resistant to L. monocytogenes, while TSCYTs are not. Like T. gondii, TSSYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell-derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.IMPORTANCECongenital toxoplasmosis can cause a devastating consequence to the fetus. To reach the fetus's tissues, Toxoplasma gondii must cross the placenta barrier. However, how this parasite crosses the placenta and the precise molecular mechanisms of placental resistance to this parasite are still unknown. In this study, we aimed to characterize a new cellular model of human trophoblast stem cells to determine their resistance, susceptibility, and response to T. gondii. Syncytiotrophoblast derived from trophoblast stem cells recapitulate the resistance profile similarly to placenta cells. We also showed that these cells are highly resistant to Listeria monocytogenes, at the level of bacterial adhesion. Our results suggest that resisting pathogen adhesion/attachment may be a generalized mechanism of syncytiotrophoblast resistance, and trophoblast stem cells represent a promising model to investigate cell-intrinsic mechanisms of resistance to pathogen adhesion and replication.

Temporal and spatial dynamics of Listeria monocytogenes central nervous system infection in mice.

Listeria monocytogenes is a bacterial pathogen that can cause life-threatening central nervous system (CNS) infections. While mechanisms by which L. monocytogenes and other pathogens traffic to the brain have been studied, a quantitative understanding of the underlying dynamics of colonization and replication within the brain is still lacking. In this study, we used barcoded L. monocytogenes to quantify the bottlenecks and dissemination patterns that lead to cerebral infection. Following intravenous (IV) inoculation, multiple independent invasion events seeded all parts of the CNS from the blood, however, only one clone usually became dominant in the brain. Sequential IV inoculations and intracranial inoculations suggested that clones that had a temporal advantage (i.e., seeded the CNS first), rather than a spatial advantage (i.e., invaded a particular brain region), were the main drivers of clonal dominance. In a foodborne model of cerebral infection with immunocompromised mice, rare invasion events instead led to a highly infected yet monoclonal CNS. This restrictive bottleneck likely arose from pathogen transit into the blood, rather than directly from the blood to the brain. Collectively, our findings provide a detailed quantitative understanding of the L. monocytogenes population dynamics that lead to CNS infection and a framework for studying the dynamics of other cerebral infections.

Synergistic and antibiofilm activity of DNase I and glucose oxidase loaded chitosan nanoparticles against dual-species biofilms of Listeria monocytogenes and Salmonella.

Salmonella and Listeria monocytogenes are two of the most common foodborne pathogens in the food industry. They form dual-species biofilms, which have a higher sensitivity to antimicrobial treatment and a greater microbial adhesion. In this experiment, we loaded DNase I and glucose oxidase (GOX) on chitosan nanoparticles (CSNPs) to explore their inhibitory effects on and disruption of dual-species biofilms of Salmonella enterica and L. monocytogenes. Transmission electron microscopy (TEM) showed that CSNP-DNase-GOX and CSNPs were spherical in shape. CSNP-DNase-GOX was shifted and altered compared to the infrared peaks of CSNPs. CSNPs loaded with DNase I and GOX showed an increase in the particle size and an alteration in the polydispersity index (PDI) and the zeta potential. Compared to free DNase I or GOX, DNase I and GOX loaded on CSNPs had higher stability at different temperatures. CSNP-DNase-GOX was more effective in inhibiting dual-species biofilms than CSNP-GOX. Scanning electron microscopy (SEM) and fluorescence microscopy were used to observe the structure of the biofilm, which further illustrated that CSNP-DNase-GOX disrupted the dual-species biofilms of S. enterica and L. monocytogenes.

Ohmic heating application with different electric fields on inactivation of Listeria monocytogenes in protein-enriched cow milk.

The aim of this study was to determine the effects of an ohmic heating (OH) process with different electric field intensities on Listeria monocytogenes inactivation in protein-enriched cow milk. Protein powder was added at rates of 2.5%, 5% and 7.5% in 1.5% fat content milk, and L. monocytogenes (ATCC 13932) strain was then inoculated into the samples. The OH process was carried out in a laboratory-type pilot unit created using stainless steel electrodes, a K-type thermocouple, a datalogger and power supply providing AC current at 0-250 V, 10 A. The inoculated milk samples were heated to 63°C by applying an electric field intensity of 10V/cm and 20V/cm. L. monocytogenes counts, pH, color measurement and hydroxymethylfurfurol levels were then determined. OH applied with an electric field intensity of 10 V/cm caused an average decrease of 5 logs in L. monocytogenes level in the samples containing 2.5% protein and decreased below the detection limit (<1 log) at the 9th minute (p<0.05). Similarly, application of an electric field intensity of 20 V/cm in milk containing 2.5% and 5% protein caused the L.monocytogenes level to decrease below the detection limit (<1 log) at 2 minutes 30 seconds (p<0.05). No change was observed in the L* (brightness) values of the samples but it was determined that there was a slight increase in pH, a* (redness) and b* (yellowness) values compared to the control group. It was observed that the inactivation of L. monocytogenes by OH depends on the duration of the OH process, protein concentration in the milk and the applied voltage gradient.

Enterococcus mundtii A2 biofilm and its anti-adherence potential against pathogenic microorganisms on stainless steel 316L.

Pathogenic bacterial biofilms present significant challenges, particularly in food safety and material deterioration. Therefore, using Enterococcus mundtii A2, known for its antagonistic activity against pathogen adhesion, could serve as a novel strategy to reduce pathogenic colonization within the food sector. This study aimed to investigate the biofilm-forming ability of E. mundtii A2, isolated from camel milk, on two widely used stainless steels within the agri-food domain and to assess its anti-adhesive properties against various pathogens, especially on stainless steel 316L. Additionally, investigations into auto-aggregation and co-aggregation were also conducted. Plate count methodologies revealed increased biofilm formation by E. mundtii A2 on 316L, followed by 304L. Scanning electron microscopy (SEM) analysis revealed a dense yet thin biofilm layer, playing a critical role in reducing the adhesion of L. monocytogenes CECT 4032 and Staphylococcus aureus CECT 976, with a significant reduction of ≈ 2 Log CFU/cm2. However, Gram-negative strains, P. aeruginosa ATCC 27853 and E. coli ATCC 25922, exhibit modest adhesion reduction (~ 0.7 Log CFU/cm2). The findings demonstrate the potential of applying E. mundtii A2 biofilms as an effective strategy to reduce the adhesion and propagation of potentially pathogenic bacterial species on stainless steel 316L.

Surveillance of pathogenic bacteria on a food matrix using machine-learning-enabled paper chromogenic arrays.

Global food systems can benefit significantly from continuous monitoring of microbial food safety, a task for which tedious operations, destructive sampling, and the inability to monitor multiple pathogens remain challenging. This study reports significant improvements to a paper chromogenic array sensor - machine learning (PCA-ML) methodology sensing concentrations of volatile organic compounds (VOCs) emitted on a species-specific basis by pathogens by streamlining dye selection, sensor fabrication, database construction, and machine learning and validation. This approach enables noncontact, time-dependent, simultaneous monitoring of multiple pathogens (Listeria monocytogenes, Salmonella, and E. coli O157:H7) at levels as low as 1 log CFU/g with over 90% accuracy. The report provides theoretical and practical frameworks demonstrating that chromogenic response, including limits of detection, depends on time integrals of VOC concentrations. The paper also discusses the potential for implementing PCA-ML in the food supply chain for different food matrices and pathogens, with species- and strain-specific identification.

Alternative splicing induced by bacterial pore-forming toxins sharpens CIRBP-mediated cell response to Listeria infection.

Cell autonomous responses to intracellular bacteria largely depend on reorganization of gene expression. To gain isoform-level resolution of these modes of regulation, we combined long- and short-read transcriptomic analyses of the response of intestinal epithelial cells to infection by the foodborne pathogen Listeria monocytogenes. Among the most striking isoform-based types of regulation, expression of the cellular stress response regulator CIRBP (cold-inducible RNA-binding protein) and of several SRSFs (serine/arginine-rich splicing factors) switched from canonical transcripts to nonsense-mediated decay-sensitive isoforms by inclusion of 'poison exons'. We showed that damage to host cell membranes caused by bacterial pore-forming toxins (listeriolysin O, perfringolysin, streptolysin or aerolysin) led to the dephosphorylation of SRSFs via the inhibition of the kinase activity of CLK1, thereby driving CIRBP alternative splicing. CIRBP isoform usage was found to have consequences on infection, since selective repression of canonical CIRBP reduced intracellular bacterial load while that of the poison exon-containing isoform exacerbated it. Consistently, CIRBP-bound mRNAs were shifted towards stress-relevant transcripts in infected cells, with increased mRNA levels or reduced translation efficiency for some targets. Our results thus generalize the alternative splicing of CIRBP and SRSFs as a common response to biotic or abiotic stresses by extending its relevance to the context of bacterial infection.

DegU-mediated suppression of carbohydrate uptake in Listeria monocytogenes increases adaptation to oxidative stress.

The foodborne bacterial pathogen Listeria monocytogenes exhibits remarkable survival capabilities under challenging conditions, severely threatening food safety and human health. The orphan regulator DegU is a pleiotropic regulator required for bacterial environmental adaptation. However, the specific mechanism of how DegU participates in oxidative stress tolerance remains unknown in L. monocytogenes. In this study, we demonstrate that DegU suppresses carbohydrate uptake under stress conditions by altering global transcriptional profiles, particularly by modulating the transcription of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS)-related genes, such as ptsH, ptsI, and hprK. Specifically, in the absence of degU, the transcripts of ptsI are significantly upregulated and those of hprK are significantly downregulated in response to copper ion-induced stress. Overexpression of ptsI significantly increases bacterial growth in vitro, while overexpression of hprK leads to a decrease in growth. We further demonstrate that DegU directly senses oxidative stress, downregulates ptsI transcription, and upregulates hprK transcription. Additionally, through an electrophoretic mobility shift assay, we demonstrate that DegU directly regulates the transcription of ptsI and hprK by binding to specific regions within their respective promoter sequences. Notably, the putative pivotal DegU binding sequence for ptsI is located from 38 to 68 base pairs upstream of the ptsH transcription start site (TSS), whereas for hprK, it is mapped from 36 to 124 base pairs upstream of the hprK TSS. In summary, we elucidate that DegU plays a significant role in suppressing carbohydrate uptake in response to oxidative stress through the direct regulation of ptsI and hprK.ImportanceUnderstanding the adaptive mechanisms employed by Listeria monocytogenes in harsh environments is of great significance. This study focuses on investigating the role of DegU in response to oxidative stress by examining global transcriptional profiles. The results highlight the noteworthy involvement of DegU in this stress response. Specifically, DegU acts as a direct sensor of oxidative stress, leading to the modulation of gene transcription. It downregulates ptsI transcription while it upregulates hprK transcription through direct binding to their promoters. Consequently, these regulatory actions impede bacterial growth, providing a defense mechanism against stress-induced damage. These findings gained from this study may have broader implications, serving as a reference for studying adaptive mechanisms in other pathogenic bacteria and aiding in the development of targeted strategies to control L. monocytogenes and ensure food safety.

Aqueous Ozone Efficacy for Inactivation of Foodborne Pathogens on Vegetables Used in Raw Meat-Based Diets for Companion Animals.

The present study evaluates the efficacy of a batch wash ozone sanitation system (BWOSS) and spray wash ozone sanitation system (SWOSS) against Listeria monocytogenes (two strains) and Salmonella enterica subsp. enterica (three serovars) inoculated on the surface of carrots, sweet potatoes, and butternut squash, commonly used in raw meat-based diets (RMBDs) marketed for companion animals such as dogs and cats. Produce either remained at room temperature for 2 h or were frozen at -20°C and then tempered overnight at 4°C to mimic the preprocessing steps of a raw pet food processing operation ('freeze-temper') prior to ozone treatment. Two ozone concentrations (0 and 5 ppm) were applied for either 20 s or 60 s for BWOSS and 20 s for SWOSS. Based on an ANOVA, BWOSS data showed no significant difference (P > 0.05) in microbial reduction between 0 and 5 ppm ozone concentration across all treatment durations for each produce type. BWOSS resulted in mean microbial reductions of up to 1.56 log CFU/mL depending on the treatment time and produce type. SWOSS data were analyzed using a generalized linear model with Quasipoisson errors. Freeze-tempered produce treated with SWOSS had a higher bacterial log reduction at 5 ppm ozone compared to 0 ppm ozone (P = 0.0013) whereas room temperature produce treated with SWOSS did not show any significant difference in microbial reduction between ozone concentrations. The potential to mitigate microbial cross-contamination was also investigated during SWOSS treatment. The results indicate that 5 ppm ozone decreased pathogens in the rinsate and proximal surfaces by 0.63-1.66 log CFU/mL greater than no ozone depending on the pathogen and sample. Overall, data from this study indicate that SWOSS would be more effective compared to BWOSS in reducing the microbial load present on the surface of root tubers and squash subjected to freezing and thawing and has the potential to mitigate cross-contamination within RMDB manufacturing environments.

ActuAtor, a Listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerization.

Form and function are often interdependent throughout biology. Inside cells, mitochondria have particularly attracted attention since both their morphology and functionality are altered under pathophysiological conditions. However, directly assessing their causal relationship has been beyond reach due to the limitations of manipulating mitochondrial morphology in a physiologically relevant manner. By engineering a bacterial actin regulator, ActA, we developed tools termed "ActuAtor" that inducibly trigger actin polymerization at arbitrary subcellular locations. The ActuAtor-mediated actin polymerization drives striking deformation and/or movement of target organelles, including mitochondria, Golgi apparatus, and nucleus. Notably, ActuAtor operation also disperses non-membrane-bound entities such as stress granules. We then implemented ActuAtor in functional assays, uncovering the physically fragmented mitochondria being slightly more susceptible to degradation, while none of the organelle functions tested are morphology dependent. The modular and genetically encoded features of ActuAtor should enable its application in studies of the form-function interplay in various intracellular contexts.

Mechanism of acid and alkali electrolyzed water on the elimination of Listeria monocytogenes biofilm based on proteomic analysis.

Acidic electrolyzed water is a relatively mature bactericide, which has a certain inhibitory effect on a variety of microorganisms, and is widely used in the field of food processing for cleaning, sterilization and disinfection. This study investigated the deactivation mechanisms of Listeria monocytogenes by Tandem Mass Tags quantitative proteomics analysis. Samples were treated through A1S4 (Alkaline electrolytic water treatment for 1 min and Acid electrolytic water treatment for 4 min), S3A1S1 (Acid electrolyzed water treatment 3 min, Alkaline electrolyzed water treatment 1 min and Acid electrolyzed water treatment 1 min), S5 (Acid electrolytic water treatment for 5 min). Proteomic analysis showed that the mechanism of acid alkaline electrolyzed water treatment to eliminate the inactivation of the biofilm of L. monocytogenes was related to protein transcription and extension, RNA processing and synthesis, gene regulation, sugar and amino acid transport and metabolism, signal transduction and ATP binding. The study on the influence mechanism and action mechanism of the combination of acidic and alkaline electrolyzed water to remove L. monocytogenes biofilm is helpful to understand the development of the process of removing biofilm by electrolyzed water, and provides theoretical support for the treatment of other microbial contamination problems in food processing by electrolyzed water.

Manganese uptake mediated by the NRAMP-type transporter MntH is required for acid tolerance in Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen that is characterized by its ability to withstand mild stresses (i.e. cold, acid, salt) often encountered in food products or food processing environments. In the previous phenotypic and genotypic characterization of a collection of L. monocytogenes strains, we have identified one strain 1381, originally obtained from EURL-lm, as acid sensitive (reduced survival at pH 2.3) and extremely acid intolerant (no growth at pH 4.9, which supports the growth of most strains). In this study, we investigated the cause of acid intolerance in strain 1381 by isolating and sequencing reversion mutants that were capable of growth at low pH (pH 4.8) to a similar extent as another strain (1380) from the same MLST clonal complex (CC2). Whole genome sequencing showed that a truncation in mntH, which encodes a homologue of an NRAMP (Natural Resistance-Associated Macrophage Protein) type Mn2+ transporter, is responsible for the acid intolerance phenotype observed in strain 1381. However, the mntH truncation alone was not sufficient to explain the acid sensitivity of strain 1381 at lethal pH values as strain 1381R1 (a mntH+ revertant) exhibited similar acid survival to its parental strain at pH 2.3. Further growth experiments demonstrated that Mn2+ (but not Fe2+, Zn2+, Cu2+, Ca2+, or Mg2+) supplementation fully rescues the growth of strain 1381 under low pH conditions, suggesting that a Mn2+ limitation is the likely cause of growth arrest in the mntH- background. Consistent with the important role of Mn2+ in the acid stress response was the finding that mntH and mntB (both encoding Mn2+ transporters) had higher transcription levels following exposure to mild acid stress (pH 5). Taken together, these results provide evidence that MntH-mediated Mn2+ uptake is essential for the growth of L. monocytogenes under low pH conditions. Moreover, since strain 1381 was recommended for conducting food challenge studies by the European Union Reference Laboratory, the use of this strain in evaluating the growth of L. monocytogenes in low pH environments where Mn2+ is scarce should be reconsidered. Furthermore, since it is unknown when strain 1381 acquired the mntH frameshift mutation, the ability of the strains used for challenge studies to grow under food-related stresses needs to be routinely validated.

Synergistic effects of 915 MHz microwave heating and essential oils on inactivation of foodborne pathogen in hot-chili sauce.

Essential oil is a food additive with antimicrobial properties but with limitations due to strong organoleptic properties. However, thermal treatments can be applied to reduce essential oil content while ensuring antimicrobial activities in food matrices. In this study, the inactivation efficiency of essential oils on E. coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in buffered peptone water (BPW) and hot-chili sauce was evaluated when coupled with 915 MHz microwave heating. Essential oils used in this study did not affect the dielectric properties and further heating rate of BPW and hot-chili sauce. The dielectric constant of BPW was 76.3 and dielectric loss factor was 30.9. In addition, it took 85 s to reach 100 °C for all samples. Among essential oils, synergistic microbial inactivation with microwave heating was observed from carvacrol (CL) and citral (CI), but not from eugenol (EU) and Carvone (CN). Specifically, CL and microwave heating (M) for 45 s showed the most effective inactivation (ca. 6 log reduction) for the pathogens in BPW. Similar trends were shown in hot-chili sauce. However, M + CI inactivation did not show synergistic effects in hot-chili sauce. Microwave heating time for hot-chilis sauce was 40 s. In propidium iodide uptake study, M + CL was found to cause most severe damage to cell membrane (758.5 of PI value for E. coli O157:H7) while M + CU and M + CN had little impact. In DiBAC4(3) test, CL resulted in the largest value (2.09 for E. coli O157:H7). These observations highlight that CL induces synergistic effects as it caused severe membrane damage along with destruction of membrane potential. The combined treatment did not show any significant difference in quality change compared to untreated hot-chili sauce (p > 0.05). The result indicates the potential application of CL and M combination for hot-chili sauce processes to ensure microbiological safety with acceptable quality.