Probiotic disruption of quorum sensing reduces virulence and increases cefoxitin sensitivity in methicillin-resistant Staphylococcus aureus.

Therapies which target quorum sensing (QS) systems that regulate virulence in methicillin-resistant Staphylococcus aureus (MRSA) are a promising alternative to antibiotics. QS systems play a crucial in the regulation of MRSA antibiotic resistance, exotoxin production, antioxidant protection and immune cell evasion, and are therefore attractive therapeutic targets to reduce the virulence of a pathogen. In the present work the the effects of bioactive peptides isolated from two strains of lactic acid bacteria were tested against antibiotic resistance, carotenoid production, resistance to oxidative killing and biofilm structure in two clinical MRSA isolates. The results obtained from fractional-inhibitory concentration assays with bulk and semi-purified bioactive molecules showed a significant synergistic effect increasing cefoxitin mediated killing of MRSA. This was coupled to a six-fold decrease of the major membrane pigment staphyloxanthin, and a 99% increase in susceptibility to oxidative stress mediated killing. Real-time quantitative PCR analysis of the QS-genes agrA and luxS, showed differential expression between MRSA strains, and a significant downregulation of the hemolysin gene hla. Light microscopy and scanning electron microscopy revealed alteration in biofilm formation and clustering behavior. These results demonstrate that bioactive metabolites may be effectively applied in tandem with beta-lactam antibiotics to sensitize MRSA to cefoxitin. Moreover, these results shown that several key QS-controlled virulence mechanisms are diminished by probiotic metabolites.

Rapid Detection of Hepatitis A Virus in Foods Using a Bioluminescent Assay in Real-Time (BART) and Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Technology.

Foodborne hepatitis A infections have been considered as a major threat for public health worldwide. Increased incidences of hepatitis A virus (HAV) infection has been associated with growing global trade of food products. Rapid and sensitive detection of HAV in foods is very essential for investigating the outbreaks. Real-time RT-PCR has been most widely used for the detection of HAV by far. However, the technology relies on fluorescence determination of the amplicon and requires sophisticated, high-cost instruments and trained personnel, limiting its use in low resource settings. In this study, a robust, affordable, and simple assay, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in combination with a bioluminescence-based determination of amplification in real-time (BART), was developed for the detection of HAV in different food matrices, including green onion, strawberry, mussel, and milk. The efficiencies of a one-step RT-LAMP-BART and a two-step RT-LAMP-BART were investigated for the detection of HAV in different food matrices and was compared with that of real-time RT-PCR. The sensitivity of the RT-LAMP-BART assay was significantly affected by Mg2+ concentration (P < 0.05), in addition to primer quality. The optimal Mg2+ concentration was 2 mM for one-step RT-LAMP-BART and 4 mM for two-step RT-LAMP-BART. Compared with cartridge-purified primers, HPLC-purified primers could greatly improve the sensitivity of the RT-LAMP-BART assay (P < 0.05). For detecting HAV in different food matrices, the performance of two-step RT-LAMP-BART was comparable with that of real-time RT-PCR and was better than that of one-step RT-LAMP-BART. The detection limit of the two-step RT-LAMP-BART for HAV in green onion, strawberry, mussel, and milk was 8.3 × 100 PFU/15 g, 8.3 × 101 PFU/50 g, 8.3 × 100 PFU/5 g, and 8.3 × 100 PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection.

Efficient capturing and sensitive detection of hepatitis A virus from solid foods (green onion, strawberry, and mussel) using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles and real-time RT-PCR.

Hepatitis A virus (HAV) continues to be a public health concern and has caused large foodborne outbreaks and economic losses worldwide. Rapid detection of HAV in foods can help to confirm the source of outbreaks in a timely manner and prevent more people getting infected. In order to efficiently detect HAV at low levels of contamination in foods, rapid and easy-to-use techniques are required to separate and concentrate viral particles to a small volume. In the current study, HAV particles were eluted from green onion, strawberry, and mussel using glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and suspended viral particles were captured using protamine-coated magnetic nanoparticles (PMNPs). This process caused a selective concentration of the viral particles, which could be followed by quantitative real-time RT-PCR analysis. Results showed that pH, NaCl concentration, and PMNP amount used for the capturing had significant effects on the recovery efficiency of HAV (P < 0.05). The highest recovery rate was obtained at pH 9.0, 0.14 M NaCl, and 50 µL of PMNPs. The optimized PMNP capturing method enabled the rapid capture and concentration of HAV. A sensitive real-time RT-PCR test was developed with detection limits of 8.3 × 100 PFU/15 g, 8.3 × 101 PFU/50 g, and 8.3 × 100 PFU/5 g of HAV in green onion, strawberry, and mussel, respectively. In conclusion, the PMNP method is rapid and convenient in capturing HAV from complex solid food samples and can generate concentrated HAV sample solutions suitable for high-sensitivity real time RT-PCR detection of the virus.

Does structure affect biological function? Modifications to the protein and phospholipids fraction of the milk fat globule membrane after extraction affect the antiproliferative activity of colon cancer cells.

In this work, the known antiproliferative activity of the untreated milk fat globule membrane (MFGM) against human colon cancer cells was employed to test the hypothesis that the supramolecular structure of the MFGM is of important biological significance. The results indicated that there is a relationship between the extent of thermal denaturation and the loss of antiproliferative capacity. There was also a clear reduction of the biological activity, when the MFGM was treated by hydrolysis using trypsin or phospholipase A2 , enzymes specific either for the protein or the phospholipids components present in the MFGM. It was concluded that the bioactivity of the MFGM can not be explained only by the presence of bioactive components, but that their structural organization plays a critical role in the antiproliferative activities of the extracts. PRACTICAL APPLICATIONS: The milk fat globule membrane (MFGM) is characterized by a complex composition and structure, with biological significance. It is known that with processing, the composition of the MFGM is modified, due to protein-protein interactions at the interface. In this work, the MFGM was isolated from untreated milk and while maintaining its overall composition, its molecular and supramolecular structures were modified using heating or specific hydrolysis to the protein or phospholipids' components. All targeted modifications affected the bioefficacy of the MFGM against colon cancer cells, thus demonstrating the importance of processing history on the functionality of the MFGM.

Corrigendum: Cell-Free Spent Media Obtained From Bifidobacterium bifidum and Bifidobacterium crudilactis Grown in Media Supplemented with 3'-Sialyllactose Modulate Virulence Gene Expression in Escherichia coli O157:H7 and Salmonella Typhimurium.

[This corrects the article DOI: 10.3389/fmicb.2016.01460.].

Concentration of hepatitis A virus in milk using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles.

Hepatitis A virus (HAV) continues to be the leading cause of viral hepatitis. HAV outbreaks have been linked to the consumption of milk, but methods for HAV detection in milk are very limited. We developed a method to concentrate HAV in milk using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles (PMNPs). In this study, protamine was covalently coated on the surface of the MNPs (20-30 nm) by a three-step chemical reaction. The successful linkage of protamine to the MNPs was confirmed by Fourier transform infrared spectroscopy (FTIR), zeta potential, and transmission electron microscopy (TEM). When used for concentrating HAV from 40 mL of milk, 50 µL of PMNPs were added to the sample and mixed for 20 min by gentle rotation, followed by a magnet capture for 30 min. The captured PMNPs were washed with glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and HAV RNA was extracted using the QIAamp MinElute Virus Spin Kit and quantified by real-time RT-PCR. The method showed a detection limit of 8.3 × 100 PFU of HAV in milk. The whole concentration procedure could be completed in approximately 50 min. The developed method was simple, inexpensive, and easy-to-perform.

Source attribution at the food sub-product level for the development of the Canadian Food Inspection Agency risk assessment model.

Decreasing the health burden caused by foodborne pathogens is challenging and it depends on the identification of the most significant hazards and food sources causing illnesses, so adequate mitigation strategies can be implemented. In this regard, the Canadian Food Inspection Agency (CFIA) has developed the Establishment-based Risk Assessment (ERA) model, so that a more effective and efficient allocation of resources can be assigned to the highest food safety risk areas. To assess risk, the model considers the type of food sub-products being manufactured by establishments and its scope is limited to the 17 most important foodborne pathogens representing the highest level of food safety risk. However, the information on source attribution at the sub-product level based on a structured approach is limited. To overcome this challenge, an expert elicitation was conducted in 2016 to estimate the relative contribution and associated certainty of each sub-product for 31 pathogen-commodity combinations to the total Canadian health burden associated with foodborne illnesses (expressed in DALYs). These DALYs represent 78% of the total Canadian health burden associated with federally-regulated food commodities considered within the model. A total of 49 Canadian experts recruited using a "snow ball" sampling strategy participated in the study by completing an electronic survey. Results of the elicitation displayed variable levels of health burden allocation between the pathogens and the different commodity sub-products. Assessment of the certainty levels showed some combinations being evaluated with more confidence (e.g., Campylobacter and eggs/poultry sub-products) than others, where a bimodal distribution of certainty was observed (e.g., Toxoplasma in pork sub-products). Furthermore, no participant raised concerns on the food classification scheme, suggesting their agreement with the proposed sub-products categorization of the elicitation. Relative contribution estimates will be included in the CFIA ERA model and used to enhance its applicability for risk prioritization and effective resource allocation during food establishment inspections. While substantial uncertainty around the central tendency estimates was found, these estimates provide a good basis for regulatory oversight and public health policy.

Immobilization of Intact Phage and Phage-Derived Proteins for Detection and Biocontrol Purposes.

The natural specificity of bacteriophages toward their hosts represents great potential for the development of platforms for the capture and detection of bacterial pathogens. Whole phage can carry reporter genes to alter the phenotype of the target pathogen. Phage can also act as staining agents or the progeny of the infection process can be detected. Alternatively, using phage components as probes offer advantages over whole phage particles, including smaller probe size and resilience to desiccation. Phage structures can be engineered for improved affinity, specificity, and binding properties. However, such concepts require the ability to anchor phage and phage-components onto mechanical supports such as beads or flat surfaces. The ability to orient the anchoring is desired in order to optimize binding efficiency. This chapter presents various methods that have been employed for the attachment of phage and phage components onto support structures such as beads, filters, and sensor surfaces.

Selection of risk factors to be included in the Canadian Food Inspection Agency risk assessment inspection model for food establishments.

The Canadian Food Inspection Agency (CFIA) is developing a risk assessment model for food establishments. Previous research on the significance of food safety risk factors determined by literature review and expert advice served as the bases for the current study, to further refine, discriminate and select the most important criteria to be included in the model. This process considered the availability of data sources, the clarity and measurability of the selected factors, undertook the elimination of lower-rated risk factors and grouped those with similar focus of attention, enabling the selection of a final list of risk factors for the model. A method of assessment for the remaining factors was then proposed to allow the quantification of individual risk factors within the model. From the 155 risk factors initially identified, 17 consolidated factors were kept and will be considered for the development of the risk assessment model.

Print to detect: a rapid and ultrasensitive phage-based dipstick assay for foodborne pathogens.

Foodborne pathogens are a burden to the economy and a constant threat to public health. The ability to rapidly detect the presence of foodborne pathogens is a vital component of any strategy towards establishing a safe and secure food supply chain. Bacteriophages (phages) are viruses capable of infecting and replicating within bacteria in a strain-specific manner. The ubiquitous and selective nature of phages makes them ideal for the detection and biocontrol of bacteria. Therefore, the objective of this research was to develop and test a phage-based paper dipstick biosensor for the detection of various foodborne pathogens in food matrices. The first step was to identify the best method for immobilizing phages on paper such that their biological activity (infectivity) was preserved. It was found that piezoelectric inkjet printing resulted in lower loss of phage infectivity when compared with other printing methods (namely gravure and blade coating) and that ColorLok paper was ideally suited to create functional sensors. The phage-based bioactive papers developed with use of piezoelectric inkjet printing actively lysed their target bacteria and retained this antibacterial activity for up to 1 week when stored at room temperature and 80% relative humidity. These bioactive paper strips in combination with quantitative real-time PCR were used for quantitative determination of target bacteria in broth and food matrices. A phage dipstick was used to capture and infect Escherichia coli O157:H7, E. coli O45:H2, and Salmonella Newport in spinach, ground beef and chicken homogenates, respectively, and quantitative real-time PCR was used to detect the progeny phages. A detection limit of 10-50 colony-forming units per millilitre was demonstrated with a total assay time of 8 h, which was the duration of a typical work shift in an industrial setting. This detection method is rapid and cost-effective, and may potentially be applied to a broad range of bacterial foodborne pathogens. Graphical abstract ᅟ.

Long-Term Preservation of Bacteriophage Antimicrobials Using Sugar Glasses.

The antimicrobial activity of LISTEX P100, Salmonella CG4, and E. coli AG10 bacteriophages were preserved in pullulan-trehalose mixture as dried films and as coatings on food packaging. The phages encapsulated in pullulan-trehalose films were able to retain infectivity for up to 3 months at ambient storage conditions. Various buffers, disaccharides and disaccharide concentrations were investigated to optimize the long-term stability of the phages in the films. It was found that pullulan and trehalose need to be simultaneously present in the film to provide the stabilizing effect and that the presence of buffers that lead to the formation of crystals in the films must be avoided for phage activity to be maintained. Overall, this study describes a method of preserving bacteriophage activity in a dried format that has great potential for use as coatings, which can be used to create antimicrobial surfaces for food preparation and for food preservation.

Molecules produced by probiotics prevent enteric colibacillosis in pigs.

BACKGROUND: With the advent of antimicrobial resistance in animal pathogens, novel methods to combat infectious diseases are being sought. Among these, probiotics have been proposed as a means of promoting animal health but problems with their use has been reported. Research has demonstrated that bioactive molecules produced during the growth of certain probiotics interfere with bacterial cell-to-cell communication, which consequently results in an attenuation of virulence in a number of pathogens, including E. coli. The objective of this study was to determine the efficacy of the bioactive molecules, termed proteobiotics, produced by Lactobacillus acidophilus in preventing enterotoxigenic E, coli (ETEC) infection in pigs, which is the etiological agent for enteric colibacillosis, a common disease of nursing and young pigs. RESULTS: To achieve this, piglets were fed a preparation of the bioactive at four levels: 0, 0.5×, 1.0× and 2.0× for 7 days prior to challenge with E. coli K88. There were 36 pigs (18 gilts and 18 barrows) per treatment, resulting in 144 piglets in total for the study. Each pen had 6 piglets (3 gilts and 3 barrows). Only piglets with no physical abnormality or conditions were used in the trial and intact male piglets and ridglings were excluded. The bioactive continued to be fed to the pigs post-challenge. Based of fecal and demeanour scores, pigs fed the low and high dose of the proteobiotic were significanlty less likely to show symptoms of illness than pigs fed no bioactive. While not being significant, the weight gain of pigs given the proteobiotics was improved. At day 4 following challenge, almost 50% of piglets that did not receive the proteobiotic were shedding ETEC in their feces, compared with about 15% of animals receiving the supplement. There was also an indication that the proteobiotics reduced colonization of the ileum by E. coli K88 and improved gut health. CONCLUSION: This study indicates that the bioactive molecules produced by L. acidophilus reduces incidence of enteric colibacillosis in pigs and their use on farms would help to reduce antibiotic use.

Seeds of the Wild Progenitor of Maize Possess Bacteria That Antagonize Foodborne Pathogens.

Endophytes are microorganisms that inhabit plant tissues without causing disease. Some endophytes help their hosts to combat pathogens. Here we explored the hypothesis that the plant-derived foods consumed by humans and other animals host endophytes that also antagonize foodborne pathogens or food-rotting agents. Our laboratory previously cultured a library of bacterial endophytes from different members of the maize/corn family (Zea) including wild relatives. Here, 190 of these endophytes were screened for their ability to antagonize four foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Clostridium perfringens, and Salmonella enterica Newport) and a food spoiling agent (Pseudomonas fluorescens) using dual culture assays. Two Paenibacillus polymyxa endophytes (strains 3C6 and 3G11) were found to inhibit the growth of all five deleterious strains on agar. Using conserved polymerase chain reaction primers and sequencing, both beneficial endophytes were found to encode polymyxin genes, suggesting a potential antibacterial mechanism of action. Polymyxin production by both strains was confirmed using enzyme-linked immunosorbent assay. Strains 3C6 and 3G11 originated, respectively, from the seeds of the wild Central American maize species Zea diploperennis, and the wild ancestor of modern maize, Zea mays ssp parviglumis (Parviglumis). As the latter is the direct ancestor of modern maize, we discuss the role its endophyte(s) may have played in promoting crop domestication by suppressing foodborne pathogens and/or food-spoilage agents.

Cell-Free Spent Media Obtained from Bifidobacterium bifidum and Bifidobacterium crudilactis Grown in Media Supplemented with 3'-Sialyllactose Modulate Virulence Gene Expression in Escherichia coli O157:H7 and Salmonella Typhimurium.

Complex oligosaccharides from human milk (HMO) possess an antimicrobial activity and can promote the growth of bifidobacteria such as Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis. In addition, fermentation of carbohydrates by bifidobacteria can result in the production of metabolites presenting an antivirulence effect on several pathogenic bacteria. Whey is rich in complex bovine milk oligosaccharides (BMO) structurally similar to HMO and B. crudilactis, a species of bovine origin, is able to metabolize some of those complex carbohydrates. This study focused on the ability of B. bifidum and B. crudilactis to grow in a culture medium supplemented in 3'-sialyllactose (3'SL) as the main source of carbon, a major BMO encountered in cow milk. Next, the effects of cell-free spent media (CFSM) were tested against virulence expression of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Both strains were able to grow in presence of 3'SL, but B. crudilactis showed the best growth (7.92 ± 0.3 log cfu/ml) compared to B. bifidum (6.84 ± 0.9 log cfu/ml). Then, CFSM were tested for their effects on virulence gene expression by ler and hilA promoter activity of luminescent mutants of E. coli and S. Typhimurium, respectively, and on wild type strains of E. coli O157:H7 and S. Typhimurium using RT-qPCR. All CFSM resulted in significant under expression of the ler and hilA genes for the luminescent mutants and ler (ratios of -15.4 and -8.1 respectively) and qseA (ratios of -2.1 and -3.1) for the wild type strain of E. coli O157:H7. The 3'SL, a major BMO, combined with some bifidobacteria strains of bovine or human origin could therefore be an interesting synbiotic to maintain or restore the intestinal health of young children. These effects observed in vitro will be further investigated regarding the overall phenotype of pathogenic agents and the exact nature of the active molecules.

Temporal distribution of encapsulated bacteriophages during passage through the chick gastrointestinal tract.

Encapsulation of bacteriophages ("phage") protects phage against environmental deactivation and provides a product that is easy to handle for storage and application with animal feed as an antibiotic alternative. The objective of this study was to evaluate an orally administered, encapsulated phage for efficient phage release in the gastrointestinal tract (GIT) of young chicks receiving feed. An optimized formulation that consisted of 0.8% low molecular weight (MW) alginate, 2% ultra-low molecular weight alginate and 3% whey protein completely released the encapsulated phage within 60 min under simulated intestinal conditions. This product was given to broiler chicks to determine passage time and distribution of the viable phage within the GIT. Following a single oral dose of 109 plaque-forming unit (PFU)/chick, the major portion (peak concentration) of the encapsulated phage passed through the chick's GIT and was detected in the feces within 4 h, with low levels being continuously excreted for up to 24 h. In comparison, the passage of free phage through the GIT occurred faster as indicated by a peak concentration in feces after 1.5 h. In assessing the temporal phage distribution, both encapsulated and free phage treatments showed no apparent difference, both having low levels of 102 to 106 PFU/g of contents along the entire GIT after 1, 2 and 4 h. These low concentrations recovered in vivo led us to examine various exposure conditions (with feed, fecal material, and buffer solutions) that were suspected to have affected phage viability/infectivity during oral delivery, sample recovery, and enumeration by plaque assay. Results showed that the exposure conditions examined did not significantly reduce phage viability and could not account for the observed low phage levels following oral administration in chicks that are on feed. In conclusion, an oral encapsulated phage dose can take more than 4 h to completely move through the GIT of young chicks. Thus, repeated or higher doses may be necessary to attain higher phage concentrations in the GIT.

Yersinia enterocolitica-Specific Infection by Bacteriophages TG1 and ϕR1-RT Is Dependent on Temperature-Regulated Expression of the Phage Host Receptor OmpF.

UNLABELLED: Bacteriophages present huge potential both as a resource for developing novel tools for bacterial diagnostics and for use in phage therapy. This potential is also valid for bacteriophages specific for Yersinia enterocolitica To increase our knowledge of Y. enterocolitica-specific phages, we characterized two novel yersiniophages. The genomes of the bacteriophages vB_YenM_TG1 (TG1) and vB_YenM_ϕR1-RT (ϕR1-RT), isolated from pig manure in Canada and from sewage in Finland, consist of linear double-stranded DNA of 162,101 and 168,809 bp, respectively. Their genomes comprise 262 putative coding sequences and 4 tRNA genes and share 91% overall nucleotide identity. Based on phylogenetic analyses of their whole-genome sequences and large terminase subunit protein sequences, a genus named Tg1virus within the family Myoviridae is proposed, with TG1 and ϕR1-RT (R1RT in the ICTV database) as member species. These bacteriophages exhibit a host range restricted to Y. enterocolitica and display lytic activity against the epidemiologically significant serotypes O:3, O:5,27, and O:9 at and below 25°C. Adsorption analyses of lipopolysaccharide (LPS) and OmpF mutants demonstrate that these phages use both the LPS inner core heptosyl residues and the outer membrane protein OmpF as phage receptors. Based on RNA sequencing and quantitative proteomics, we also demonstrate that temperature-dependent infection is due to strong repression of OmpF at 37°C. In addition, ϕR1-RT was shown to be able to enter into a pseudolysogenic state. Together, this work provides further insight into phage-host cell interactions by highlighting the importance of understanding underlying factors which may affect the abundance of phage host receptors on the cell surface. IMPORTANCE: Only a small number of bacteriophages infecting Y. enterocolitica, the predominant causative agent of yersiniosis, have been previously described. Here, two newly isolated Y. enterocolitica phages were studied in detail, with the aim of elucidating the host cell receptors required for infection. Our research further expands the repertoire of phages available for consideration as potential antimicrobial agents or as diagnostic tools for this important bacterial pathogen.

Development of prototypes of bioactive packaging materials based on immobilized bacteriophages for control of growth of bacterial pathogens in foods.

Due to lack of adequate control methods to prevent contamination in fresh produce and growing consumer demand for natural products, the use of bacteriophages has emerged as a promising approach to enhance safety of these foods. This study sought to control Listeria monocytogenes in cantaloupes and RTE meat and Escherichia coli O104:H4 in alfalfa seeds and sprouts under different storage conditions by using specific lytic bacteriophage cocktails applied either free or immobilized. Bacteriophage cocktails were introduced into prototypes of packaging materials using different techniques: i) immobilizing on positively charged modified cellulose membranes, ii) impregnating paper with bacteriophage suspension, and iii) encapsulating in alginate beads followed by application of beads onto the paper. Phage-treated and non-treated samples were stored for various times and at temperatures of 4°C, 12°C or 25°C. In cantaloupe, when free phage cocktail was added, L. monocytogenes counts dropped below the detection limit of the plating technique (<1 log CFU/g) after 5 days of storage at both 4°C and 12°C. However, at 25°C, counts below the detection limit were observed after 3 and 6h and a 2-log CFU/g reduction in cell numbers was seen after 24h. For the immobilized Listeria phage cocktail, around 1-log CFU/g reduction in the Listeria count was observed by the end of the storage period for all tested storage temperatures. For the alfalfa seeds and sprouts, regardless of the type of phage application technique (spraying of free phage suspension, bringing in contact with bacteriophage-based materials (paper coated with encapsulated bacteriophage or impregnated with bacteriophage suspension)), the count of E. coli O104:H4 was below the detection limit (<1 log CFU/g) after 1h in seeds and about a 1-log cycle reduction in E. coli count was observed on the germinated sprouts by day 5. In ready-to-eat (RTE) meat, LISTEX™ P100, a commercial phage product, was able to significantly reduce the growth of L. monocytogenes at both storage temperatures, 4°C and 10°C, for 25 days regardless of bacteriophage application format (immobilized or non-immobilized (free)). In conclusion, the developed phage-based materials demonstrated significant antimicrobial effect, when applied to the artificially contaminated foods, and can be used as prototypes for developing bioactive antimicrobial packaging materials capable of enhancing the safety of fresh produce and RTE meat.

Lactococcus lactis subsp. cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro.

Lactococcus lactis subsp. cremoris JFR1 has been studied in reduced fat cheese due to its ability to produce exopolysaccharides (EPS) in situ, contributing to improved textural and organoleptic properties. In this study, the effect of strain JFR1 on virulence gene expression and attachment of Salmonella to HT-29 human colon carcinoma cells was investigated. Overnight cultures of L. lactis subsp. cremoris JFR1 containing EPS, grown in M17 media with 0.5% glucose supplementation, decreased attachment as well as down regulated virulence gene expression in Salmonella enterica subsp. enterica when tested on HT-29 cells. However, EPS isolated from milk fermented with L. lactis subsp. cremoris JFR1 did not affect Salmonella virulence gene expression or attachment to HT-29 cells. These results suggest that EPS does not contribute to the attachment of Salmonella to human intestinal cells. However, the possibility that the isolation process may have affected the structural features of EPS cannot be ruled out.

Inhibitory Effect of Epigallocatechin Gallate on the Virulence of Clostridium difficile PCR Ribotype 027.

Clostridium difficile infection (CDI) is the most prevalent cause of health-care-associated infections. CDI-related health-care costs and deaths are both increasing annually on a global scale. C. difficile have been reported in food products in Canada, Europe, and the United States; however, the systematic transmission of C. difficile between humans and animals is yet to be understood. Because of the limitations of current therapeutic options, there is a need for the development of new patient treatments. Epigallocatechin gallate (EGCG) is a major catechin compound found in green tea extracts and exhibits antioxidant and antimicrobial activities. This study was conducted to investigate the inhibitory effects of EGCG on the expression of virulence genes in C. difficile and in C. difficile-associated diseases by inhibition of quorum sensing. The protein expression of autoinducer-2 (AI-2) was evaluated by AI-2 activity. EGCG at various concentrations had an inhibitory effect on AI-2 production, especially at 10 µg/mL. EGCG also significantly repressed the transcription of virulence genes, including luxS and tcdA, and prolonged the survival of Caenorhabditis elegans infected with C. difficile. Furthermore, treatment with EGCG effectively protected C. difficile-infected mice from C. difficile-induced death. Histological analysis of the colon and cecum of these mice revealed that EGCG protected tissues of the lower intestinal tract from damage. EGCG exerted growth-inhibitory and bactericidal activities on C. difficile in C. difficile-infected mice. Our results suggest that EGCG has significant antipathogenic effects on C. difficile and can be used to prevent or treat C. difficile-associated diseases or C. difficile infections.