Dopamine receptor D2 confers colonization resistance via microbial metabolites.

The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.

Thermal Inactivation of Escherichia Phage OSYSP and Host Strain Escherichia coli O157:H7 EDL933: A Comparative Kinetic Analysis.

Lytic bacteriophages are promising biocontrol agents against pathogenic bacteria for food and therapeutic applications. Investigating the feasibility of combining phage and physical lethal agents, such as heat, as an effective hurdle combination could lead to beneficial applications. The current research was initiated to compare the thermal inactivation kinetics of a lytic phage (Escherichia phage OSYSP) and its host (Shiga toxin-producing Escherichia coli O157:H7 EDL933), considering they have different critical thermal targets in their structures. To provide a basis for comparison, thermal inactivation kinetics were determined on suspensions of these agents in buffered peptone water using a thermally controlled circulating water bath. Results showed that the bacteriophage virions have a remarkable heat resistance (p < 0.05) compared to their host cells. The D-values of the populations of phage (PFU/mL) and EDL933 strain (CFU/mL) were 166.7 and 7.3 min at 55°C, compared to 44.4 and 0.3 min at 60°C, respectively. Additionally, D-values were significantly (p < 0.05) more influenced by temperature changes in the case of E. coli O157:H7 EDL933 (z-value 3.7°C) compared to that for phage OSYSP (z-value 7.7°C). When the phage suspension was heat-treated in a thermal cycler instead of a water bath, no significant differences between the two treatment procedures (p > 0.05) in estimating virus D- and z-values were observed. Based on these findings, it may be feasible to combine phage OSYSP with mild heat during processing of food to selectively inactivate E. coli O157:H7 EDL933 and subsequently maintain product safety during storage by the surviving phage population; however, the feasibility of this application needs to be investigated. Additionally, the relatively heat-resistant phage OSYSP could qualify as a biological indicator to validate thermal treatments of minimally processed foods in which E. coli O157:H7 EDL933 is the pathogen-of-concern.

Bacterial outer membrane vesicles provide an alternative pathway for trafficking of Escherichia coli O157 type III secreted effectors to epithelial cells.

IMPORTANCE: Bacteria can package protein cargo into nanosized membrane blebs that are shed from the bacterial membrane and released into the environment. Here, we report that a type of pathogenic bacteria called enterohemorrhagic Escherichia coli O157 (EHEC) uses their membrane blebs (outer membrane vesicles) to package components of their type 3 secretion system and send them into host cells, where they can manipulate host signaling pathways including those involved in infection response, such as immunity. Usually, EHEC use a needle-like apparatus to inject these components into host cells, but packaging them into membrane blebs that get taken up by host cells is another way of delivery that can bypass the need for a functioning injection system.

Acid adaptation increased the resistance of Escherichia coli O157:H7 in bok choy (Brassica rapa subsp. chinensis) juice to high-pressure processing.

IMPORTANCE: Based on the U.S. Food and Drug Administration regulations, E. coli O157:H7 is a pertinent pathogen in high acid juices that needs to be inactivated during the pasteurization process. The results of this study suggest that the effect of acid adaptation should be considered in the selection of HPP parameters for E. coli O157:H7 inactivation to ensure that pasteurization objectives are achieved.

Socializing at the Air-Liquid Interface: a Functional Genomic Analysis on Biofilm-Related Genes during Pellicle Formation by Escherichia coli and Its Interaction with Aeromonas australiensis.

Pellicles are biofilms that form at the air-liquid interface. We demonstrated that specific strains of Escherichia coli formed pellicles in single cultures when cocultured with Carnobacterium maltaromaticum and E. coli O157:H7 but not with Aeromonas australiensis. Therefore, a combination of comparative genomic, mutational, and transcriptome analyses were applied to identify the unique genes in pellicle formation and investigate gene regulation under different growth phases. Here, we report that pellicle-forming strains do not harbor unique genes relative to non-pellicle-forming strains; however, the expression level of biofilm-related genes differed, especially for the genes encoding curli. Further, the regulatory region of curli biosynthesis is phylogenetically different among pellicle- and non-pellicle-forming strains. The disruption on modified cellulose and regulatory region of curli biosynthesis abolished pellicle formation in strains of E. coli. Besides, the addition of quorum sensing molecules (C4-homoserine lactones [C4-HSL]), synthesized by Aeromonas species, to pellicle formers abolished pellicle formation and implied a role of quorum sensing on pellicle formation. The deletion of autoinducer receptor sdiA in E. coli did not restore pellicle formation when cocultured with A. australiensis but modulated expression level of genes for curli and cellulose biosynthesis, resulting in a thinner layer of pellicle. Taken together, this study identified genetic determinants for pellicle formation and characterized the switching between pellicle to surface-associated biofilm in a dual-species environment, facilitating better understanding of the mechanisms for pellicle formation in E. coli and related organisms. IMPORTANCE To date, most attention has focused on biofilm formation on solid surfaces. By comparison, the knowledge on pellicle formation at the air-liquid interface is more limited and few studies document how bacteria decide on whether to form biofilms on solid surfaces or pellicles at the air-liquid interface to the surface-associated biofilms at the bottom. In this report, we characterized the regulation of biofilm-related genes during pellicle formation and document that interspecies communication via quorum sensing contributes to regulating the switch from pellicle to surface-associated biofilm. The discoveries expand the current view of regulatory cascades associated with pellicle formation.

Carvacrol-loaded nanoemulsions produced with a natural emulsifier for lettuce sanitization.

Carvacrol is an antimicrobial agent that shows potential for eliminating microorganisms in vegetables, increasing food safety. However, intense odor and low water solubility of carvacrol are limiting factors for its application for fresh vegetables sanitization, which can be overcome by nanotechnology. Two different nanoemulsions containing carvacrol (11 mg/mL) were developed by probe sonication: carvacrol-saponin nanoemulsion (CNS) and carvacrol-polysorbate 80 nanoemulsion (CNP). Formulations presented appropriate droplet sizes (from 74.7 nm to 168.2 nm) and high carvacrol encapsulation efficiency (EE) (from 89.5 % to 91.5 %). CNS showed adequate droplet size distribution (PDI < 0.22) and high zeta potential values (around -30 mV) compared to CNP, with saponin chosen for the following experiments. Carvacrol nanoemulsions presented Bacterial Inactivation Concentration (BIC) against the Salmonella cocktail from 5.51 to 0.69 mg/mL and for the E. coli cocktail from 1.84 to 0.69 mg/mL. Among all tested nanoemulsions, CNS1 presented the lowest BIC (0.69 mg/mL) against both bacterial cocktails. Damage to bacterial cells in lettuce treated with nanoemulsion was confirmed by scanning electron microscopy. For lettuce sanitization, CNS1 showed a similar effect to unencapsulated carvacrol, with a high bacterial reduction (>3 log CFU/g) after lettuce immersion for 15 min at 2 × BIC. Using the same immersion time, the CNS1 (2 × BIC) demonstrated equal or better efficacy in reducing both tested bacterial cocktails (>3 log CFU/g) when compared to acetic acid (6.25 mg/mL), citric acid (25 mg/mL), and sodium hypochlorite solution (150 ppm). Lettuce immersed in CNS1 at both concentrations (BIC and 2 × BIC) did not change the color and texture of leaves, while the unencapsulated carvacrol at 2 × BIC darkened them and reduced their firmness. Consequently, carvacrol-saponin nanoemulsion (CNS1) proved to be a potential sanitizer for lettuce.

Migration risk of Escherichia coli O157:H7 in unsaturated porous media in response to different colloid types and compositions.

The vadose zone is a critical zone for microbial entry into the subsurface environment, and various types of inorganic and organic colloids can affect the migration of pathogenic bacteria. In the study, we explored the migration behavior of Escherichia coli O157:H7 with humic acids (HA), iron oxides (Fe2O3) or their mixture, uncovering their migration mechanisms in the vadose zone. The effect of complex colloids on the physiological properties of E. coli O157:H7 was analyzed based on the measured particle size, zeta potential and contact angle. HA colloids significantly promoted the migration of E. coli O157:H7, where Fe2O3 was opposite. The migration mechanism of E. coli O157:H7 with HA and Fe2O3 is obviously different. Multiple colloids dominated by organic colloid will further highlight its promoting effect on E. coli O157:H7 under the guidance of electrostatic repulsion due to the influence of colloidal stability. Multiple colloids dominated by metallic colloid will inhibit the migration of E. coli O157:H7 under the control of capillary force due to the restriction of contact angle. The risk of secondary release of E. coli O157:H7 can be effectively reduced when the ratio of HA/Fe2O3 is ≥ 1. Combining this conclusion with the distribution characteristics of soil in China, an attempt was made to analyse the migration risk of E. coli O157:H7 on a national scale. In China, from north to south, the migration capacity of E. coli O157:H7 gradually decreased, and the risk of secondary release gradually increased. These results provide ideas for the subsequent study of the effect of other factors on the migration of pathogenic bacteria on a national scale and provide risk information about soil colloids for the construction of pathogen risk assessment model under comprehensive conditions in the future.

Human cathelicidin LL-37 exerts amelioration effects against EHEC O157:H7 infection regarding inflammation, enteric dysbacteriosis, and impairment of gut barrier function.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection impairs intestinal barrier function, causing intestinal inflammation and enteric dysbacteriosis. The human cathelicidin LL-37 can regulate excessive inflammatory responses, barrier function, and balance the intestinal microbial community; however, little is known about its effects on inflammation, intestinal barrier function, and microbiota disorders in EHEC O157:H7-infected mice. In this study, we investigated the protective effect of LL-37 against EHEC O157:H7 infection and elucidated the underlying mechanism using a mouse model. LL-37 treatment was found to inhibit body weight loss, restore edema and destruction of the intestinal villi, and significantly reduce epithelial apoptosis (P < 0.05) in EHEC O157:H7-infected mice. Furthermore, inflammatory infiltration of macrophages and neutrophils into the jejunum and colon was significantly decreased (P < 0.05). LL-37 significantly downregulated the production of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) (P < 0.05) and upregulated the anti-inflammatory cytokine (IL-10) during EHEC O157:H7 infection. LL-37 increased the expression of tight junction proteins (ZO-1, ZO-2, claudin-1, and occludin), which are associated with intestinal barrier function, and had a positive effect on EHEC O157:H7-induced microbial disorders, particularly in terms of the inflammation-related microbiota. LL-37 also significantly decreased the E. coli load in the liver and spleen (P < 0.01) and restored the structure of the liver and kidney. Taken together, LL-37 conferred protection in a EHEC O157:H7-induced mouse model by reducing intestinal inflammation, enhancing intestinal barrier function, and restoring the balance of the intestinal microbiota, which indicates the therapeutic potential of LL-37 against pathogen infection.

Unraveling enterohemorrhagic Escherichia coli infection: the promising role of dietary compounds and probiotics in bacterial elimination and host innate immunity boosting.

The innate immune system has developed sophisticated strategies to defense against infections. Host cells utilize the recognition machineries such as toll-like receptors and nucleotide binding and oligomerization domain-like receptors to identify the pathogens and alert immune system. However, some pathogens have developed tactics to evade host defenses, including manipulation of host inflammatory response, interference with cell death pathway, and highjack of phagocytosis signaling for a better survival and colonization in host. Enterohemorrhagic Escherichia coli (EHEC) is a notorious foodborne pathogen that causes severe tissue damages and gastrointestinal diseases, which has been reported to disturb host immune responses. Diverse bioactive compounds such as flavonoids, phenolic acids, alkaloids, saccharides, and terpenoids derived from food varieties and probiotics have been discovered and investigated for their capability of combating bacterial infections. Some of them serve as novel antimicrobial agents and act as immune boosters that harness host immune system. In this review, we will discuss how EHEC, specifically E. coli O157:H7, hijacks the host immune system and interferes with host signaling pathway; and highlight the promising role of food-derived bioactive compounds and probiotics in harnessing host innate immunity and eliminating E. coli O157:H7 infection with multiple strategies.

Transcriptomic analysis reveals the antibacterial mechanism of phenolic compounds from kefir fermented soy whey against Escherichia coli 0157:H7 and Listeria monocytogenes.

Transcriptomic analysis was used to investigate the antibacterial mechanism of phenolic compounds from kefir fermented soy whey (FSP) against Escherichia coli 0157:H7 and Listeria monocytogenes. The kefir fermentation increased the concentration of several phenolic aglycones with proven antibacterial efficacy in the FSP. The time-kill curve showed that 2× MICs of the FSP killed >99.9 % of the strains within 2 h of exposure. The checkerboard fractional inhibition concentration (FIC) assay proved that phenolics were the sole antibacterial agent in the FSP. The transmission electron microscope (TEM) photomicrograph corroborated the propidium iodide (PI) uptake, protein, and nucleic acid leakage assays. They demonstrated that the phenolics permeated the cell membrane, disrupted the cytoplasm, and caused cell lysis in the treated cells leading to protein and nucleic acid leakage. The transcriptome analysis revealed that exposure of the cells to MICs of the phenolics induced molecular responses leading to differential expression of 1850 genes in E. coli 0157:H7 and 2090 in L. monocytogenes. The phenolics suppressed the expression of genes crucial for carbohydrate utilization, transmembrane glucose transport, tricarboxylic acid (TCA), and ATP synthesis. The phenolic-induced stress also downregulated the expression of quorum sensing and virulence-related genes, peptidoglycan and phospholipid synthases, and ABC transporters. The cells initiated a resistance response by stimulating the two-component signal transduction systems to trigger the over-expression of a cascade of genes involved in stress resistance, gluconeogenesis, ATPase activity and proton transmembrane transport. Nonetheless, the data indicated that the phenolics suppressed the expression of translational proteins that would have facilitated the resistance and repair of the cell damage caused by the phenolics. The study provides discrete data evidence that FSP could be used to control the pathogenicity and the proliferation of E. coli 0157:H7 and L. monocytogenes in our foods and food systems.

L. monocytogens exhibited less cell membrane damage, lipid peroxidation, and intracellular reactive oxygen species accumulation after plasma-activated water treatment compared to E. coli O157:H7 and S. Typhimurium.

This study investigated the bactericidal activity of plasma-activated water (PAW) generated with a remote discharge reactor against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes. PAW-40, -80, and -120, prepared by activating distilled water for 40, 80, and 120 min, respectively, showed inactivation activity against pathogenic bacteria, which increased as the activation time increased due to decrease in pH and increase in oxidation-reduction potential and reactive oxygen/nitrogen species (RONS) of PAW. In addition, Gram-positive bacteria L. monocytogenes showed superior resistance to PAW than Gram-negative bacteria E. coli O157:H7 and S. Typhimurium. Compared with E. coli O157:H7 and S. Typhimurium, L. monocytogens exhibited less cell membrane damage, lipid peroxidation, and intracellular ROS accumulation after PAW treatment, which indicated that L. monocytogenes exhibited greater resistance because the thick cell wall buffered RONS diffusion into the cell. PAW also showed a control effect on the pathogenic bacteria on cherry tomato, and the effect was maintained throughout five repeated applications; thus, proposing high reusability of PAW. The results of this study propose that PAW generated with a remote discharge reactor can be utilized for pathogen control and provides basic data for related research and practical industrial applications.

Heat sensitization of Escherichia coli by the natural antimicrobials vanillin and emulsified citral in blended carrot-orange juice.

Efforts have been focusing on the way to overcome the impact of heating on food quality while achieving the desired shelf life. In this sense, the thermosensitization of E. coli using the natural antimicrobials vanillin (V; 0.8 and 1.0 g/L) or/and emulsified citral (C; 0.012 and 0.025 g/L) was assessed at 58 and 60 °C in blended carrot-orange juice (pH 4.0; 9.0°Brix). All combined treatments exceeded the inactivation achieved by the single thermal treatments in half the time. The inactivation of the binary treatments (V or C + heating) at 58 °C was 3.84-0.62 log-cycles more effective than the control, particularly with vanillin. Ternary treatments (V + C + heating) at 58 °C increased the microbial reduction approximately 30%; however, at 60 °C no further inactivation was observed, suggesting the thermal effect prevailed. This was verified by the higher b Weibullian parameter and the narrower frequency distributions. The selected treatments 1.0 V + 0.012C at 58 and 60 °C were challenged against the pathogenic E. coli O157:H7 and found to be effective. Additionally, the microbiota of the juice was maintained at acceptable levels during storage (4 °C). In conclusion, there was an increase in the heat sensitivity of E. coli due to the natural antimicrobials, particularly vanillin at 58 °C. Therefore, reducing the intensity of the thermal processing will lead to clean label, high-quality juices, while addressing food safety requirements.

Novel reusable animal model for comparative evaluation of in vivo growth and protein-expression of Escherichia coli O157 strains in the bovine rumen.

Previously, we had demonstrated that Escherichia coli O157:H7 (O157) strain 86-24 expresses proteins involved in survival rather than virulence in vitro in rumen fluid from dairy cattle limit fed a maintenance diet. Here, we verified if this observation would be true for different O157 strains grown in vitro in rumen fluid from, and in vivo in the rumen of, animals on contrasting maintenance (high fiber) and lactation (high energy-protein) diets usually limit fed to dairy cattle. For the in vivo studies, an economical, novel, reusable and non-terminal rumen-fistulated animal model permitting simultaneous evaluation of multiple bacterial strains in the bovine rumen was developed. All experiments were conducted in duplicate using different animals to account for host-related variations. The O157 strains included, 86-24, EDL933 and the super shed SS-17. E. coli NalR (#5735), derived from a bovine intestinal commensal E. coli, was included as a control. As expected, diet influenced ruminal pH and volatile fatty acid (VFA) composition. The pH ranged from 6.2-7.0 and total VFA concentrations from 109-141 µM/ml, in animals fed the maintenance diet. In comparison, animals fed the lactation diet had a ruminal pH ranging between 5.18-6.0, and total VFA of 125-219 µM/ml. Strain dependent differences in O157 recovery from the rumen fluid of cattle fed either diet was observed, both in vitro and in vivo, with O157 strains 86-24 and EDL933 demonstrating similar survival patterns. Analysis of the O157 proteomes expressed in the rumen fluid/rumen verified previous observations of adaptive responses. Any difference in the adaptive response was mainly influenced by the animal's diet and growth conditions (in vitro and in vivo) and not the O157 strain. These new insights into the O157 responses could help formulate modalities to control O157 across strains in cattle at all stages of husbandry.

Enhanced high hydrostatic pressure lethality in acidulated raw pet food formulations was pathogen species and strain dependent.

Feeding dogs and cats with raw meat-based pet food is taking relevance in the recent years. The high aw of these products together with the no cooking before its consumption by the animal pose a risk due to the potential occurrence and growth of foodborne pathogens. High pressure processing (HPP) is a non-thermal emerging technology that can be used as a lethality treatment to inactivate microorganisms with a minimum impact on the sensory and nutritional traits of the product. The purpose of the present study was to evaluate the variability in pressure resistance of different strains of the relevant foodborne pathogens Salmonella spp., Escherichia coli and Listeria monocytogenes in raw pet food formulated without and with lactic acid. In general, Salmonella and L.monocytogenes strains showed a higher resistance to HPP than E. coli strains. In lactic acid acidulated formulations, the susceptibility to HPP of L. monocytogenes was markedly enhanced. The resistance to HPP was not only dependent on the microorganism but also on the strain. Thus, the selection of the proper strains should be taken into account when designing and validating the application of HPP as a control measure within the HACCP plan.

Optimization of Multivalent Gold Nanoparticle Vaccines Eliciting Humoral and Cellular Immunity in an In Vivo Model of Enterohemorrhagic Escherichia coli O157:H7 Colonization.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 remains a pathogen of significance and high consequence around the world. This outcome is due in part to the high economic impact associated with massive, contaminated product recalls, prevalence of the pathogen in carrier reservoirs, disease sequelae, and mortality associated with several outbreaks worldwide. Furthermore, the contraindication of antibiotic use for the treatment of EHEC-related infections makes this pathogen a primary candidate for the development of effective prophylactic vaccines. However, no vaccines are approved for human use, and many have failed to provide a high degree of efficacy or broad protection, thereby opening an avenue for the use of new technologies to produce a safe, effective, and protective vaccine. Building on our previous studies using reverse vaccinology-predicted antigens, we refine a formulation, evaluate new immunogenic antigens, and further expand our understanding about the mechanism of EHEC vaccine-mediated protection. In the current study, we exploit the use of the nanotechnology platform based on gold nanoparticles (AuNP), which can act as a scaffold for the delivery of various antigens. Our results demonstrate that a refined vaccine formulation incorporating EHEC antigen LomW, EscC, LpfA1, or LpfA2 and delivered using AuNPs can elicit robust antigen-specific cellular and humoral responses associated with reduced EHEC colonization in vivo. Furthermore, our in vitro mechanistic studies further support that antibody-mediated protection is primarily driven by inhibition of bacterial adherence onto intestinal epithelial cells and by promotion of macrophage uptake and killing. IMPORTANCE Enterohemorrhagic E. coli O157:H7 remains an important human pathogen that does not have an effective and safe vaccine available. We have made outstanding progress in the identification of novel protective antigens that have been incorporated into the gold nanoparticle platform and used as vaccines. In this study, we have refined our vaccine formulations to incorporate multiple antigens and further define the mechanism of antibody-mediated protection, including one vaccine that promotes macrophage uptake. We further define the cell-mediated responses elicited at the mucosal surface by our nanovaccine formulations, another key immune mechanism linked to protection.

Control of Escherichia coli Serotype O157:H7 Motility and Biofilm Formation by Salicylate and Decanoate: MarA/SoxS/Rob and pchE Interactions.

Prophage-encoded Escherichia coli O157:H7 transcription factor (TF), PchE, inhibits biofilm formation and attachment to cultured epithelial cells by reducing curli fimbriae expression and increasing flagella expression. To identify pchE regulators that might be used in intervention strategies to reduce environmental persistence or host infections, we performed a computational search of O157:H7 strain PA20 pchE promoter sequences for binding sites used by known TFs. A common site shared by MarA/SoxS/Rob TFs was identified and the typical MarA/Rob inducers, salicylate and decanoate, were tested for biofilm and motility effects. Sodium salicylate, a proven biofilm inhibitor, but not sodium decanoate, strongly reduced O157:H7 biofilms by a pchE-independent mechanism. Both salicylate and decanoate enhanced O157:H7 motility dependent on pchE using media and incubation temperatures optimum for culturing human epithelial cells. However, induction of pchE by salicylate did not activate the SOS response. MarA/SoxS/Rob inducers provide new potential agents for controlling O157:H7 interactions with the host and its persistence in the environment. IMPORTANCE There is a need to develop E. coli serotype O157:H7 nonantibiotic interventions that do not precipitate the release and activation of virulence factor-encoded prophage and transferrable genetic elements. One method is to stimulate existing regulatory pathways that repress bacterial persistence and virulence genes. Here we show that certain inducers of MarA and Rob have that ability, working through both pchE-dependent and pschE-independent pathways.

Comparison of Nonthermal Decontamination Methods to Improve the Safety for Raw Beef Consumption.

ABSTRACT: The object of this study was to examine nonthermal treatments to reduce foodborne pathogens in beef that is consumed raw. Foodborne illness pathogens (Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes) were inoculated in the raw beef eye round. Death rates of foodborne illness pathogens were evaluated by nonthermal decontamination methods: high pressure processing (HPP) at 500 MPa for 2, 5, and 7 min; UV light-emitting diode (LED) radiation at 405 nm for 2, 6, and 24 h; hypochlorous acid water (HAW) at 100 ppm for 1, 3, and 5 min; 2.5% lactic acid (LA) for 1, 3, and 5 min; modified atmosphere packaging that replaced O2 to CO2 for 24 and 48 h with anaerogen (O2 levels were less than 1% and CO2 levels were 9 to 13%); and bio-gel application (BGA) for 24 and 48 h. For the bio-gel preparation, 5% sodium alginate was dissolved in 40 mL of glycerol and mixed with 0.2% CaCl2 dissolved in 60 mL of water, and this mixture was left at room temperature for solidification. Quality characteristics (color, pH, water activity, and texture) were measured after applying the practical nonthermal decontamination application. After HPP treatment for 7 min, inactivity rates were 4.4 to 6.7 Log CFU/g (100.0%) for E. coli, Salmonella, and L. monocytogenes and 1.7 Log CFU/g (98.0%) for S. aureus (P < 0.05). After treatment with UV LED for 24 h, reduced cell counts were 0.5 Log CFU/g (67.3%), 0.7 Log CFU/g (82.2%), and 0.3 Log CFU/g (47.1%) for E. coli, Salmonella, and S. aureus, respectively (P < 0.05), but no significant reduction of 0.0 Log CFU/g (4.3%) was observed for L. monocytogenes. When the beef was treated with HAW for 5 min, 0.6 Log CFU/g (73.3%) of E. coli, 0.5 Log CFU/g (66.2%) of Salmonella, 0.4 Log CFU/g (60.7%) of S. aureus, and 0.5 Log CFU/g (65.6%) of L. monocytogenes were inactivated. After the beef was treated with LA for 5 min, 1.8 Log CFU/g (98.5%) of E. coli, 3.0 Log CFU/g (99.9%) of Salmonella, 1.3 Log CFU/g (95.4%) of S. aureus, and 1.9 Log CFU/g (98.6%) of L. monocytogenes were inactivated. Modified atmosphere packaging for 48 h caused the inactivation of 0.3 Log CFU/g (51.8%) of E. coli and 0.1 Log CFU/g (19.2%) of Salmonella. After BGA treatment for 48 h, 0.3 Log CFU/g (55.2%) of E. coli and 0.4 Log CFU/g (58.7%) of Salmonella were significantly decreased (P < 0.05). HPP cooked the beef after 2 min of treatment. HAW and BGA changed the surface color of the beef, and LA reduced the pH of beef (P < 0.05). However, UV LED did not cause changes in the beef quality properties. These results indicates that UV LED can improve the food safety of raw beef without changes in beef quality.

A Novel Nano-Antimicrobial Polymer Engineered with Chitosan Nanoparticles and Bioactive Peptides as Promising Food Biopreservative Effective against Foodborne Pathogen E. coli O157-Caused Epithelial Barrier Dysfunction and Inflammatory Responses.

For food quality and safety issues, the emergence of foodborne pathogenic bacteria has further accelerated the spread of antibiotic residues and drug resistance genes. To alleviate the harm caused by bacterial infections, it is necessary to seek novel antimicrobial agents as biopreservatives to prevent microbial spoilage. Nanoantimicrobials have been widely used in the direct treatment of bacterial infections. CNMs, formed by chitosan nanoparticles and peptides, are promising antibiotic alternatives for use as excellent new antibacterial drugs against pathogenic bacteria. Herein, the current study evaluated the function of CNMs in the protection of foodborne pathogen Escherichia coli (E. coli) O157 infection using an intestinal epithelial cell model. Antibacterial activity assays indicated that CNMs exerted excellent bactericidal activity against E. coli O157. Assessment of the cytotoxicity risks toward cells demonstrated that 0.0125-0.02% of CNMs did not cause toxicity, but 0.4% of CNMs caused cytotoxicity. Additionally, CNMs did not induced genotoxicity either. CNMs protected against E. coli O157-induced barrier dysfunction by increasing transepithelial electrical resistance, decreasing lactate dehydrogenase and promoting the protein expression of occludin. CNMs were further found to ameliorate inflammation via modulation of tumor factor α, toll-like receptor 4 and nuclear factor κB (NF-κB) expression via inhibition of mitogen-activated protein kinase and NF-κB activation and improved antioxidant activity. Taken together, CNMs could protect the host against E. coli O157-induced intestinal barrier damage and inflammation, showing that CNMs have great advantages and potential application as novel antimicrobial polymers in the food industry as food biopreservatives, bringing new hope for the treatment of bacterial infections.

Characterization of a novel phage depolymerase specific to Escherichia coli O157:H7 and biofilm control on abiotic surfaces.

The increasing prevalence of foodborne diseases caused by Escherichia coli O157:H7 as well as its ability to form biofilms poses major threats to public health worldwide. With increasing concerns about the limitations of current disinfectant treatments, phage-derived depolymerases may be used as promising biocontrol agents. Therefore, in this study, the characterization, purification, and application of a novel phage depolymerase, Dpo10, specifically targeting the lipopolysaccharides of E. coli O157, was performed. Dpo10, with a molecular mass of 98 kDa, was predicted to possess pectate lyase activity via genome analysis and considered to act as a receptor-binding protein of the phage. We confirmed that the purified Dpo10 showed O-polysaccharide degrading activity only for the E. coli O157 strains by observing its opaque halo. Dpo10 maintained stable enzymatic activities across a wide range of temperature conditions under 55°C and mild basic pH. Notably, Dpo10 did not inhibit bacterial growth but significantly increased the complement-mediated serum lysis of E. coli O157 by degrading its O-polysaccharides. Moreover, Dpo10 inhibited the biofilm formation against E. coli O157 on abiotic polystyrene by 8-fold and stainless steel by 2.56 log CFU/coupon. This inhibition was visually confirmed via fieldemission scanning electron microscopy. Therefore, the novel depolymerase from E. coli siphophage exhibits specific binding and lytic activities on the lipopolysaccharide of E. coli O157 and may be used as a promising anti-biofilm agent against the E. coli O157:H7 strain.

Transcriptional analysis reveals specific niche factors and response to environmental stresses of enterohemorrhagic Escherichia coli O157:H7 in bovine digestive contents.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) are responsible for severe diseases in humans, and the ruminant digestive tract is considered as their main reservoir. Their excretion in bovine feces leads to the contamination of foods and the environment. Thus, providing knowledge of processes used by EHEC to survive and/or develop all along the bovine gut represents a major step for strategies implementation. RESULTS: We compared the transcriptome of the reference EHEC strain EDL933 incubated in vitro in triplicate samples in sterile bovine rumen, small intestine and rectum contents with that of the strain grown in an artificial medium using RNA-sequencing (RNA-seq), focusing on genes involved in stress response, adhesion systems including the LEE, iron uptake, motility and chemotaxis. We also compared expression of these genes in one digestive content relative to the others. In addition, we quantified short chain fatty acids and metal ions present in the three digestive contents. RNA-seq data first highlighted response of EHEC EDL933 to unfavorable physiochemical conditions encountered during its transit through the bovine gut lumen. Seventy-eight genes involved in stress responses including drug export, oxidative stress and acid resistance/pH adaptation were over-expressed in all the digestive contents compared with artificial medium. However, differences in stress fitness gene expression were observed depending on the digestive segment, suggesting that these differences were due to distinct physiochemical conditions in the bovine digestive contents. EHEC activated genes encoding three toxin/antitoxin systems in rumen content and many gene clusters involved in motility and chemotaxis in rectum contents. Genes involved in iron uptake and utilization were mostly down-regulated in all digestive contents compared with artificial medium, but feo genes were over-expressed in rumen and small intestine compared with rectum. The five LEE operons were more expressed in rectum than in rumen content, and LEE1 was also more expressed in rectum than in small intestine content. CONCLUSION: Our results highlight various strategies that EHEC may implement to survive in the gastrointestinal environment of cattle. These data could also help defining new targets to limit EHEC O157:H7 carriage and shedding by cattle.