Listeria adhesion protein orchestrates caveolae-mediated apical junctional remodeling of epithelial barrier for Listeria monocytogenes translocation.

The cellular junctional architecture remodeling by Listeria adhesion protein-heat shock protein 60 (LAP-Hsp60) interaction for Listeria monocytogenes (Lm) passage through the epithelial barrier is incompletely understood. Here, using the gerbil model, permissive to internalin (Inl) A/B-mediated pathways like in humans, we demonstrate that Lm crosses the intestinal villi at 48 h post-infection. In contrast, the single isogenic (lap- or ΔinlA) or double (lap-ΔinlA) mutant strains show significant defects. LAP promotes Lm translocation via endocytosis of cell-cell junctional complex in enterocytes that do not display luminal E-cadherin. In comparison, InlA facilitates Lm translocation at cells displaying apical E-cadherin during cell extrusion and mucus expulsion from goblet cells. LAP hijacks caveolar endocytosis to traffic integral junctional proteins to the early and recycling endosomes. Pharmacological inhibition in a cell line and genetic knockout of caveolin-1 in mice prevents LAP-induced intestinal permeability, junctional endocytosis, and Lm translocation. Furthermore, LAP-Hsp60-dependent tight junction remodeling is also necessary for InlA access to E-cadherin for Lm intestinal barrier crossing in InlA-permissive hosts. IMPORTANCE: Listeria monocytogenes (Lm) is a foodborne pathogen with high mortality (20%-30%) and hospitalization rates (94%), particularly affecting vulnerable groups such as pregnant women, fetuses, newborns, seniors, and immunocompromised individuals. Invasive listeriosis involves Lm's internalin (InlA) protein binding to E-cadherin to breach the intestinal barrier. However, non-functional InlA variants have been identified in Lm isolates, suggesting InlA-independent pathways for translocation. Our study reveals that Listeria adhesion protein (LAP) and InlA cooperatively assist Lm entry into the gut lamina propria in a gerbil model, mimicking human listeriosis in early infection stages. LAP triggers caveolin-1-mediated endocytosis of critical junctional proteins, transporting them to early and recycling endosomes, facilitating Lm passage through enterocytes. Furthermore, LAP-Hsp60-mediated junctional protein endocytosis precedes InlA's interaction with basolateral E-cadherin, emphasizing LAP and InlA's cooperation in enhancing Lm intestinal translocation. This understanding is vital in combating the severe consequences of Lm infection, including sepsis, meningitis, encephalitis, and brain abscess.

Protocol to reveal the binding partner of secreted housekeeping enzymes in Listeria monocytogenes via in silico prediction to in vivo validation.

Numerous interacting protein partners exist without recognized interactive domains, necessitating a standardized methodology to decipher more in-depth interaction profiles. Here, we present a protocol to reveal the binding partner of a secreted housekeeping enzyme, alcohol acetaldehyde dehydrogenase (Listeria adhesion protein), in Listeria monocytogenes through in silico modeling and in vivo experiments. We describe steps for target protein modeling, biophysical profiling, ClusPro docking optimization, protein variant modeling, and docking comparison. We then provide detailed procedures for in vitro and in vivo protein binding validation. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Physicochemical characterization of changes in pea protein as the result of cold extrusion.

Pea protein is a popular plant-based protein for mimicking textures in meat and dairy analogues which are more sustainable than their animal-based counterparts. However, precise mechanisms for generating specific textures through different processing methods are still being evaluated. This work utilizes a novel low-temperature extrusion process to selectively alter the chemical structure of pea protein. Changes in secondary structure, surface hydrophobicity, electrostatic interactions, and disulfide bonding are characterized through FTIR, ANS- probes, zeta potential, and SDS-PAGE. Extrudates are further characterized using texture parameter analysis. It was found that a linear combination of physicochemical data, generated with multiple linear regression modelling, led to reasonable estimates of the specific mechanical energy and textural properties. This work offers a new method of reactive extrusion to selectively modify interactions in pea protein using low temperature extrusion, and applications may include fatty textures, since the extrudates are found to be largely stabilized through hydrophobic interactions evaluated with surface hydrophobicity measurements.

Human Salmonellosis: A Continuous Global Threat in the Farm-to-Fork Food Safety Continuum.

Salmonella is one of the most common zoonotic foodborne pathogens and a worldwide public health threat. Salmonella enterica is the most pathogenic among Salmonella species, comprising over 2500 serovars. It causes typhoid fever and gastroenteritis, and the serovars responsible for the later disease are known as non-typhoidal Salmonella (NTS). Salmonella transmission to humans happens along the farm-to-fork continuum via contaminated animal- and plant-derived foods, including poultry, eggs, fish, pork, beef, vegetables, fruits, nuts, and flour. Several virulence factors have been recognized to play a vital role in attaching, invading, and evading the host defense system. These factors include capsule, adhesion proteins, flagella, plasmids, and type III secretion systems that are encoded on the Salmonella pathogenicity islands. The increased global prevalence of NTS serovars in recent years indicates that the control approaches centered on alleviating the food animals' contamination along the food chain have been unsuccessful. Moreover, the emergence of antibiotic-resistant Salmonella variants suggests a potential food safety crisis. This review summarizes the current state of the knowledge on the nomenclature, microbiological features, virulence factors, and the mechanism of antimicrobial resistance of Salmonella. Furthermore, it provides insights into the pathogenesis and epidemiology of Salmonella infections. The recent outbreaks of salmonellosis reported in different clinical settings and geographical regions, including Africa, the Middle East and North Africa, Latin America, Europe, and the USA in the farm-to-fork continuum, are also highlighted.

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis.

Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42 nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.

Sequestration of zearalenone using microorganisms blend in vitro.

Zearalenone (ZEN) is an estrogenic mycotoxin produced by the Fusarium species and induces severe reproductive disorders in animals thus a major concern in the livestock industry. Probiotic bacteria treatments have been shown to inactivate mycotoxins, therefore, in this study, we investigated the effect of two commercial probiotic feed additives on the sequestration of ZEN. Commercial probiotic blends containing clay-based binder with Aspergillus niger, Bacillus licheniformis, Bacillus pumilus, and Bacillus subtilis at various proportions from BioMatrix International were incubated with ZEN in a time-dependent manner and then analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) to quantify unbound ZEN. Sequestration of ZEN was further verified by using MCF-7 cell-based cytotoxicity and/or cell proliferation assays. ZEN, or probiotic mix, was nontoxic to MCF-7 cells. Probiotic blends decreased ZEN concentration by 45% (∼100 µg L-1) and prevented ZEN from inducing MCF-7 cell proliferation (20%-28% reduction). The probiotic feed supplements tested show a potential utility in ZEN neutralization.

Bioinformatic Approaches for Characterizing Molecular Structure and Function of Food Proteins.

Structural bioinformatics analyzes protein structural models with the goal of uncovering molecular drivers of food functionality. This field aims to develop tools that can rapidly extract relevant information from protein databases as well as organize this information for researchers interested in studying protein functionality. Food bioinformaticians take advantage of millions of protein amino acid sequences and structures contained within these databases, extracting features such as surface hydrophobicity that are then used to model functionality, including solubility, thermostability, and emulsification. This work is aided by a protein structure-function relationship framework, in which bioinformatic properties are linked to physicochemical experimentation. Strong bioinformatic correlations exist for protein secondary structure, electrostatic potential, and surface hydrophobicity. Modeling changes in protein structures through molecular mechanics is an increasingly accessible field that will continue to propel food science research.

Studies on Simultaneous Enrichment and Detection of Escherichia coli O157:H7 during Sample Shipment.

The USDA-FSIS has zero tolerance for E. coli O157:H7 in raw ground beef. Currently, FSIS collects samples from beef processing facilities and ships them overnight to regional testing laboratories. Pathogen detection requires robust methods that employ an initial 15-24 h culture enrichment. This study assessed the potential of using the ΦV10nluc phage-based luminescence detection assay during enrichment while the sample is in transit. Parameters including phage concentrations, temperature, and media-to-sample ratios were evaluated. Results in liquid media showed that 1.73× 103 pfu/mL of ΦV10nluc was able to detect 2 CFU in 10 h. The detection of E. coli O157:H7 was further evaluated in kinetic studies using ratios of 1:3, 1:2, and 1:1 ground beef sample to enrichment media, yielding positive results for as little as 2-3 CFU in 325 g ground beef in about 15 h at 37 °C. These results suggest that this approach is feasible, allowing the detection of a presumptive positive upon arrival of the sample to the testing lab. As the current cargo hold controlled temperature is required to be 15-25 °C, the need for elevated temperature should be easily addressed. If successful, this approach could be expanded to other pathogens and foods.

Bacteriophage-Based Biosensors: A Platform for Detection of Foodborne Bacterial Pathogens from Food and Environment.

Foodborne microorganisms are an important cause of human illness worldwide. Two-thirds of human foodborne diseases are caused by bacterial pathogens throughout the globe, especially in developing nations. Despite enormous developments in conventional foodborne pathogen detection methods, progress is limited by the assay complexity and a prolonged time-to-result. The specificity and sensitivity of assays for live pathogen detection may also depend on the nature of the samples being analyzed and the immunological or molecular reagents used. Bacteriophage-based biosensors offer several benefits, including specificity to their host organism, the detection of only live pathogens, and resistance to extreme environmental factors such as organic solvents, high temperatures, and a wide pH range. Phage-based biosensors are receiving increasing attention owing to their high degree of accuracy, specificity, and reduced assay times. These characteristics, coupled with their abundant supply, make phages a novel bio-recognition molecule in assay development, including biosensors for the detection of foodborne bacterial pathogens to ensure food safety. This review provides comprehensive information about the different types of phage-based biosensor platforms, such as magnetoelastic sensors, quartz crystal microbalance, and electrochemical and surface plasmon resonance for the detection of several foodborne bacterial pathogens from various representative food matrices and environmental samples.

Low-Cost Nonreversible Electronic-Free Wireless pH Sensor for Spoilage Detection in Packaged Meat Products.

Contamination of meat with pathogenic microorganisms can cause severe illnesses and food waste, which has significant negative impacts on both general health and the economy. In many cases, the expiration date is not a good indicator of meat freshness as there is a high risk of contamination during handling throughout the supply chain. Many biomarkers, including color, odor, pH, temperature, and volatile compounds, are used to determine spoilage. Among these, pH presents a simple and effective biomarker directly linked to the overgrowth of bacteria and degradation of the meat tissue. Low-cost methods for wireless pH monitoring are crucial in detecting spoilage on a large commercial scale. Existing technologies are often limited to short-range detection, with the use of batteries and different electronic components that increases both the manufacturing complexity and cost of the final device. To address these shortcomings, we have developed a cost-effective wireless pH sensor, which uses passive resonant frequency (RF) sensing, combined with a pH-responsive polymer that can be placed within packaged meat products and provide a remote assessment of the risk of microbial spoilage throughout the supply chain. The sensor tag consists of a sensing resonator coated with a pH-sensitive material and a passivated reference resonator operating in a differential frequency configuration. Upon exposure to elevated pH levels >6.8, the coating on the sensing resonator dissolves, which in turn results in a distinct change in the resonant frequency with respect to the reference resonator. Systematic theoretical and experimental results at different pH levels demonstrated that a 20% shift in resonant frequency demarcates the point for spoilage detection. As a proof of concept, the performance of the sensor in remotely detecting the risk of food spoilage was validated in packaged poultry over 10 days. The sensor fabrication process takes advantage of recent developments in the scalable manufacturing of flexible, low-cost devices, including selective laser etching of metalized plastic films and doctor-blade coating of stimuli-responsive polymer films. Furthermore, the biocompatibility of all the materials used in the sensor was confirmed with human intestinal cells (HCT-8 cells).

Inactivation of Polymicrobial Biofilms of Foodborne Pathogens Using Epsilon Poly-L-Lysin Conjugated Chitosan Nanoparticles.

A mixed culture (polymicrobial) biofilm provides a favorable environment for pathogens to persist in the food processing environment and to contaminate food products. Inactivation and eradication of such biofilms from food processing environments are achieved by using harsh disinfectants, but their toxicity and environmentally hostile characteristics are unsustainable. This study aims to use food-grade natural nanoparticulated antimicrobials to control mixed-culture biofilms. Chitosan, a natural broad-spectrum antimicrobial biopolymer (polysaccharide) from crustaceans, was derivatized to produce chitosan nanoparticles (ChNP) as a carrier for another broad-spectrum antimicrobial agent, ε-poly-L-lysine (PL), to synthesize ChNP-PL conjugate. The antimicrobial activity of ChNP and ChNP-PL was tested against mixed-culture biofilms. ChNP-PL (~100 nm) exhibited a synergistic antimicrobial and anti-biofilm effect against mono or mixed-culture biofilms of five foodborne pathogens, including Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica serovar Enteritidis, Escherichia coli O157:H7, and Pseudomonas aeruginosa. ChNP-PL treatment prevented biofilm formation by mono or mixed cultures of L. monocytogenes, P. aeruginosa, and E. coli O157:H7, and bacterial counts were either below the detection limit or caused 3.5-5 log reduction. ChNP-PL also inactivated preformed biofilms. In monoculture biofilm, ChNP-PL treatment reduced L. monocytogenes counts by 4.5 logs, S. Enteritidis by 2 logs, E. coli by 2 logs, and S. aureus by 0.5 logs, while ChNP-PL had no inhibitory effect on P. aeruginosa. In vitro mammalian cell-based cytotoxicity analysis confirmed ChNP-PL to have no deleterious effect on intestinal HCT-8 cell line. In conclusion, our results show ChNP-PL has strong potential to prevent the formation or inactivation of preformed polymicrobial biofilms of foodborne pathogens.

Alginate-based antimicrobial coating reduces pathogens on alfalfa seeds and sprouts.

Foodborne illness associated with the consumption of contaminated sprouts has been a significant food safety risk. While seed disinfection with chemical sanitizers has been used as an intervention approach, its efficacy to reduce bacterial load has not been always satisfactory. In this study, a newly developed alginate-based, antimicrobial seed coating treatment was evaluated for its efficacy to reduce foodborne pathogens from alfalfa seeds and sprouts. Alfalfa seeds were inoculated with Listeria monocytogenes or Salmonella enterica serovar Typhimurium, air-dried, and thereafter subjected to different treatments. The treated seeds were analyzed for bacterial cell populations over 28 days of storage. Meanwhile, treated seeds after one day of storage were sprouted for three days and the populations of pathogens on sprouts were determined. The results showed that the alginate coating in the presence of lactic acid (alginate coating/LA) reduced both pathogens to an undetectable level one day after the treatment. With sprouts, alginate coating/LA resulted in significantly lower (P < 0.05) populations of Listeria and Salmonella than chlorine or lactic acid treatment. While the germination rate of seeds was reduced due to the use of lactic acid, the impact of alginate coating on germination was not significant. In general, this study indicated the effect of alginate coating on reducing the bacterial load from alfalfa seeds and sprouts, and further study is needed to select antimicrobial compounds and coating materials to reduce the adverse impact on germination rate.

Petri-plate, bacteria, and laser optical scattering sensor.

Classical microbiology has paved the path forward for the development of modern biotechnology and microbial biosensing platforms. Microbial culturing and isolation using the Petri plate revolutionized the field of microbiology. In 1887, Julius Richard Petri invented possibly the most important tool in microbiology, the Petri plate, which continues to have a profound impact not only on reliably isolating, identifying, and studying microorganisms but also manipulating a microbe to study gene expression, virulence properties, antibiotic resistance, and production of drugs, enzymes, and foods. Before the recent advances in gene sequencing, microbial identification for diagnosis relied upon the hierarchal testing of a pure culture isolate. Direct detection and identification of isolated bacterial colonies on a Petri plate with a sensing device has the potential for revolutionizing further development in microbiology including gene sequencing, pathogenicity study, antibiotic susceptibility testing , and for characterizing industrially beneficial traits. An optical scattering sensor designated BARDOT (bacterial rapid detection using optical scattering technology) that uses a red-diode laser, developed at the beginning of the 21st century at Purdue University, some 220 years after the Petri-plate discovery can identify and study bacteria directly on the plate as a diagnostic tool akin to Raman scattering and hyperspectral imaging systems for application in clinical and food microbiology laboratories.

Bacterial Biofilms and Their Implications in Pathogenesis and Food Safety.

Biofilm formation is an integral part of the microbial life cycle in nature. In food processing environments, bacterial transmissions occur primarily through raw or undercooked foods and by cross-contamination during unsanitary food preparation practices. Foodborne pathogens form biofilms as a survival strategy in various unfavorable environments, which also become a frequent source of recurrent contamination and outbreaks of foodborne illness. Instead of focusing on bacterial biofilm formation and their pathogenicity individually, this review discusses on a molecular level how these two physiological processes are connected in several common foodborne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica and Escherichia coli. In addition, biofilm formation by Pseudomonas aeruginosa is discussed because it aids the persistence of many foodborne pathogens forming polymicrobial biofilms on food contact surfaces, thus significantly elevating food safety and public health concerns. Furthermore, in-depth analyses of several bacterial molecules with dual functions in biofilm formation and pathogenicity are highlighted.

Antibody- and nucleic acid-based lateral flow immunoassay for Listeria monocytogenes detection.

Listeria monocytogenes is an invasive opportunistic foodborne pathogen and its routine surveillance is critical for protecting the food supply and public health. The traditional detection methods are time-consuming and require trained personnel. Lateral flow immunoassay (LFIA), on the other hand, is an easy-to-perform, rapid point-of-care test and has been widely used as an inexpensive surveillance tool. In recent times, nucleic acid-based lateral flow immunoassays (NALFIA) are also developed to improve sensitivity and specificity. A significant improvement in lateral flow-based assays has been reported in recent years, especially the ligands (antibodies, nucleic acids, aptamers, bacteriophage), labeling molecules, and overall assay configurations to improve detection sensitivity, specificity, and automated interpretation of results. In most commercial applications, LFIA has been used with enriched food/environmental samples to ensure detection of live cells thus prolonging the assay time to 24-48 h; however, with the recent improvement in LFIA sensitivity, results can be obtained in less than 8 h with shortened and improved enrichment practices. Incorporation of surface-enhanced Raman spectroscopy and/or immunomagnetic separation could significantly improve LFIA sensitivity for near-real-time point-of-care detection of L. monocytogenes for food safety and public health applications.

Listeria monocytogenes: review of pathogenesis and virulence determinants-targeted immunological assays.

Listeria monocytogenes is one of the most invasive foodborne pathogens and is responsible for numerous outbreaks worldwide. Most of the methods to detect this bacterium in food require selective enrichment using traditional bacterial culture techniques that can be time-consuming and labour-intensive. Moreover, molecular methods are expensive and need specific technical knowledge. In contrast, immunological approaches are faster, simpler, and user-friendly alternatives and have been developed for the detection of L. monocytogenes in food, environmental, and clinical samples. These techniques are dependent on the constitutive expression of L. monocytogenes antigens and the specificity of the antibodies used. Here, updated knowledge on pathogenesis and the key immunogenic virulence determinants of L. monocytogenes that are used for the generation of monoclonal and polyclonal antibodies for the serological assay development are summarised. In addition, immunological approaches based on enzyme-linked immunosorbent assay, immunofluorescence, lateral flow immunochromatographic assays, and immunosensors with relevant improvements are highlighted. Though the sensitivity and specificity of the assays were improved significantly, methods still face many challenges that require further validation before use.

Current State of Development of Biosensors and Their Application in Foodborne Pathogen Detection.

ABSTRACT: Foodborne disease outbreaks continue to be a major public health and food safety concern. Testing products promptly can protect consumers from foodborne diseases by ensuring the safety of food before retail distribution. Fast, sensitive, and accurate detection tools are in great demand. Therefore, various approaches have been explored recently to find a more effective way to incorporate antibodies, oligonucleotides, phages, and mammalian cells as signal transducers and analyte recognition probes on biosensor platforms. The ultimate goal is to achieve high specificity and low detection limits (1 to 100 bacterial cells or piconanogram concentrations of toxins). Advancements in mammalian cell-based and bacteriophage-based sensors have produced sensors that detect low levels of pathogens and differentiate live from dead cells. Combinations of biotechnology platforms have increased the practical utility and application of biosensors for detection of foodborne pathogens. However, further rigorous testing of biosensors with complex food matrices is needed to ensure the utility of these sensors for point-of-care needs and outbreak investigations.

Effects of fulvic acid size on microcystin-LR photodegradation and detoxification in the chlorine/UV process.

Microcystin-LR (MC-LR), a polypeptide toxin generated by cyanobacteria, threatens the safety of drinking water supplies. In this study, fulvic acid (FA) was separated into two molecular weight (MW) ranges to evaluate the effects of FA size on MC-LR degradation in the chlorine/UV process. The rates of MC-LR degradation were significantly reduced in FA-containing water (3.7 × 10-3 s-1 for small MW FA; 4.3 × 10-3 s-1 for large MW FA) as compared with FA free water (4.9 × 10-3 s-1). The contributions of ClO• to MC-LR degradation were dramatically lower in small MW FA water (0.4%) than large MW FA (13.9%) and FA free water (17.4%), suggesting inhibition by lignin-like substances in FA in the transformation of Cl• to ClO• and scavenging ClO•. Monochlorination and hydroxylation occurred in the first step of the MC-LR degradation process. The accumulation of intermediate products in the chlorine/UV process indicated that small MW FA inhibited further degradation of MC-LR. Small MW FA, rather than MC-LR degradation, was the dominant factor in minimizing MC-LR cytotoxicity toward a human intestinal epithelial cell line.

Biofilm-isolated Listeria monocytogenes exhibits reduced systemic dissemination at the early (12-24 h) stage of infection in a mouse model.

Environmental cues promote microbial biofilm formation and physiological and genetic heterogeneity. In food production facilities, biofilms produced by pathogens are a major source for food contamination; however, the pathogenesis of biofilm-isolated sessile cells is not well understood. We investigated the pathogenesis of sessile Listeria monocytogenes (Lm) using cell culture and mouse models. Lm sessile cells express reduced levels of the lap, inlA, hly, prfA, and sigB and show reduced adhesion, invasion, translocation, and cytotoxicity in the cell culture model than the planktonic cells. Oral challenge of C57BL/6 mice with food, clinical, or murinized-InlA (InlAm) strains reveals that at 12 and 24 h post-infection (hpi), Lm burdens are lower in tissues of mice infected with sessile cells than those infected with planktonic cells. However, these differences are negligible at 48 hpi. Besides, the expressions of inlA and lap mRNA in sessile Lm from intestinal content are about 6.0- and 280-fold higher than the sessle inoculum, respectively, suggesting sessile Lm can still upregulate virulence genes shortly after ingestion (12 h). Similarly, exposure to simulated gastric fluid (SGF, pH 3) and intestinal fluid (SIF, pH 7) for 13 h shows equal reduction in sessile and planktonic cell counts, but induces LAP and InlA expression and pathogenic phenotypes. Our data show that the virulence of biofilm-isolated Lm is temporarily attenuated and can be upregulated in mice during the early stage (12-24 hpi) but fully restored at a later stage (48 hpi) of infection. Our study further demonstrates that in vitro cell culture assay is unreliable; therefore, an animal model is essential for studying the pathogenesis of biofilm-isolated bacteria.