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Invasion of Salmonella into host intestinal epithelial cells requires the expression of virulence genes. In this study, cell culture models of human intestinal cells (mucus-producing HT29-MTX cells, absorptive Caco-2 cells, and combined cocultures of the two) were used to determine the effects of Lactococcus lactis subsp. cremoris treatments (exopolysaccharide producing and nonproducing strains) on the virulence gene expression of Salmonella Typhimurium and its mutant lacking the oligopeptide permease subunit A (ΔoppA). During the course of epithelial cell (HT29-MTX, Caco-2, and combined) infection by Salmonella Typhimurium DT104, improved barrier function was reflected by increased transepithelial electrical resistance in cells treated with both strains of L. lactis subsp. cremoris. In addition, virulence gene expression was downregulated, accompanied with lower numbers of invasive bacteria into epithelial cells in the presence of L. lactis subsp. cremoris treatments. Similarly, virulence gene expression of Salmonella was also suppressed when coincubated with overnight cultures of both L. lactis subsp. cremoris strains in the absence of epithelial cells. However, in medium or in the presence of cell cultures, Salmonella lacking the OppA permease function remained virulent. HT29-MTX cells and combined cultures stimulated by Salmonella Typhimurium DT104 showed significantly lower secretion levels of pro-inflammatory cytokine IL-8 after treatment with L. lactis subsp. cremoris cell suspensions. Contrarily, these responses were not observed during infection with S. Typhimurium ΔoppA. Both the exopolysaccharide producing and nonproducing strains of L. lactis subsp. cremoris JFR1 exhibited an antivirulence effect against S. Typhimurium DT104 although no significant difference between the two strains was observed. Our results show that an intact peptide transporter is essential for the suppression of Salmonella virulence genes which leads to the protection of the barrier function in the cell culture models studied.

The Canadian Food Inspection Agency (CFIA), as part of the modernization of its inspection system, is developing a risk assessment model with the goal of evaluating the most important food safety-related risk factors associated with the various types of food establishments under its jurisdiction. Ultimately, this new risk assessment model will assist CFIA in allocating its inspection resources according to the level of risk associated with each establishment. As a first step, key risk factors need to be identified. The objective of the current study was thus to identify and evaluate the importance of food safety-related risk factors that could be included in a risk assessment model. An initial literature search was used to identify these risk factors. Thereafter, 75 Canadian food safety experts were asked to assess the relative importance of the 155 identified risk factors with respect to food safety. This step was carried out using a web-based questionnaire in which risk factors had to be assessed by experts using a ten-point Likert-type scale. One risk factor (“management commitment”) received a median score of 10, while median score of 9, 8.5 and 8, and below 8 were attributed to 42, 77 and 35 of the risk factors listed, respectively. A company's commitment towards food safety was identified as an important risk factor contributing to the magnitude of the risk attributed to a food establishment. Many factors related to the performance of an establishment's preventive control plans were also scored high by experts, such as sanitation, hygiene practices, calibration and employee training. The median interquartile range in risk factor scoring generally did not exceed 2, and the respondent profile (work experience, employer and/or commodity-based expertise) did not have a strong influence on score results based on Kruskal–Wallis tests. This observation suggests that, despite their different backgrounds, experts’ opinions were relatively consistent and comparable when scoring the impact of various risk factors towards food safety. These survey outcomes represent useful information to aid in the design and implementation of risk assessment models for food establishments.

Research into the connection between organizational effectiveness and culture has been documented since the early nineteen nineties. A connection between economic performance and organizational culture has been established directly linking strong cultural drivers to economic performance in both the finance and retail sectors. This research proposes a similar association between food safety culture, the measures of maturity and cost of poor quality. Through data collected at five multi-national food companies, this association is explored, and an improved food safety maturity model suggested. The authors also propose a dynamic model of food safety culture, segmenting it into 4 building blocks: I. Organizational effectiveness, II. Organizational culture norms, III. Working group learned and shared assumptions, and behaviours, and IV. Individual intent and behaviours; and discuss the crucial role of actions between building blocks as part of the pathway to realizing economic gain.

Bacteriophages have been envisioned as tools to control a variety of foodborne pathogenic bacteria. Salmonella is a foodborne pathogen that is a threat to public health around the world. Contaminated tomatoes have been associated with several Salmonella outbreaks. Hence, the objective of this work was to identify and characterize different lytic bacteriophages against Salmonella Newport, as one of top ten Salmonella serovars associated with human salmonellosis in North America, and then apply these phages to enhance the safety of cherry tomatoes. Four lytic phages against Salmonella Newport were selected based on their ability to lyse a majority of the 26 screened Salmonella serovars. The selected phages belong to Myoviridae (vB_SnwM_CGG4-1, vB_SnwM_CGG4-2) and Siphoviridae (vB_SnwM_CGG3-1, vB_SnwM_CGG3-2) families. They were found to be stable at different temperatures and pH, have latent periods ranging from 53 to 65 min and burst sizes from 92 to 177. In addition, the two Myoviridae phages have a lower frequency of developing bacteriophage insensitive mutants when compared with the Siphoviridae phages. No significant change in virulence gene expression was observed in the developed bacteriophage insensitive mutants when compared to the parental phage sensitive strain. Furthermore, the vB_SnwM_CGG4-1 genome revealed no homology to virulence or lysogenic genes. A phage cocktail was used to control the growth of S. Newport in broth medium and on contaminated cherry tomato. Complete inhibition of bacterial growth in broth medium was observed at 25 °C for 24 h. In addition, a 4.5 log10 unit reduction in the bacterial count was observed when applying the phage cocktail onto contaminated tomatoes stored at 22 °C for 3 days. These findings suggest that the isolated phages can be used for biocontrol of S. Newport to improve the safety of ready-to-eat (RTE) produce.

Clostridium difficile infection is a range of toxin - mediated intestinal diseases that is often acquired in hospitals and small communities in developed countries. The main virulence factors of C. difficile are two exotoxins, toxin A and toxin B, which damage epithelial cells and manifest as colonic inflammation and mild to severe diarrhea. Inhibiting C. difficile adherence, colonization, and reducing its toxin production could substantially minimize its pathogenicity and lead to faster recovery from the disease. This study investigated the efficacy of probiotic secreted bioactive molecules from Lactobacillus acidophilus La-5, in decreasing C. difficile attachment and cytotoxicity in human epithelial cells in vitro. L. acidophilus La-5 cell-free supernatant (La-5 CFS) was used to treat the hypervirulent C. difficile ribotype 027 culture with subsequent monitoring of cytotoxicity and adhesion. In addition, the effect of pretreating cell lines with La-5 CFS in protecting cells from the cytotoxicity of C. difficile culture filtrate or bacterial cell attachment was examined. La-5 CFS substantially reduced the cytotoxicity and cytopathic effect of C. difficile culture filtrate on HT-29 and Caco-2 cells. Furthermore, La-5 CFS significantly reduced attachment of the C. difficile bacterial cells on both cell lines. It was also found that pretreatment of cell lines with La-5 CFS effectively protected cell lines from cytotoxicity and adherence of C. difficile. Our study suggests that La-5 CFS could potentially be used to prevent and cure C. difficile infection and relapses.

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 × 10

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.

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

The Canadian Food Inspection Agency (CFIA) is developing a risk assessment model aimed at quantifying the food safety risk associated with food establishments under its jurisdiction. To support the development of this model, the current study was undertaken to quantify the relative importance of selected criteria considered for inclusion in the model. This process also aimed at estimating the risk associated with specific clusters of criteria. Overall, 173 criteria were presented to experts during a two-round face-to-face expert elicitation to estimate their relative risk to human health. Twenty-nine Canadian experts participated in the expert elicitation including members from academia (31%), industry (31%), and government (38%). A good consensus on the relative risks given to most criteria and clusters of criteria was achieved, and experts assessed them as significantly affecting the risk related to a food establishment. None of the experts expressed opposition to the inclusion of any criterion or to the way they were clustered. Experts assigned a relative risk of ≤4, of 4–8, and of ≥8–67% (116), 29.5% (51), and 3.5% (6) of the 173 criteria identified, respectively. Those having the highest impact on establishment food safety risk were: historical food safety recalls and lack of compliance for the sanitation program, the control of critical control points, followed by the equipment maintenance and calibration program, and the general food hygiene program. Having a sampling plan with trend analysis and follow-up actions was considered as an important mitigation factor. As a result, the median values calculated for each criterion and cluster will be used in the new Canadian Food Inspection Agency Establishment-based risk assessment model to support the allocation of inspection resources based on risk.

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.

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.

The innate specificity of bacteriophages toward their hosts makes them excellent candidates for the development of detection assays. They can be used in many ways to detect pathogens, and each has its own advantages and disadvantages. Whole bacteriophages can carry reporter genes to alter the phenotype of the target. Bacteriophages can act as staining agents or the progeny of the infection process can be detected, which further increases the sensitivity of the detection assay. Compared with whole-phage particles, use of phage components as probes offers other advantages: for example, smaller probe size to enhance binding activity, phage structures that can be engineered for better affinity, as well as specificity, binding properties, and robustness. When no natural binding with the target exists, phages can be used as vehicles to identify new protein-ligand interactions necessary for diagnostics. This review comprehensively summarizes many uses of phages as detection tools and points the way toward how phage-based technologies may be improved.

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 A(2), 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.

Outbreaks of enterohemorrhagic Escherichia coli O157:H7 have been commonly associated with products derived from ground beef, but recently the organism has been implicated as the causative agent in outbreaks involving yogurt and cheese. This finding has raised concern about the potential for its growth and survival in fermented dairy products. A bioluminescent strain of E. coli O157:H7 was used to determine postprocessing survival in yogurt with live cultures at pH 4.17, 4.39, and 4.47 stored at 4 and 10°C. In addition, survival of E. coli O157:H7 was monitored during the manufacture of Cottage, Colby, Romano, and Feta cheeses. Results indicated survival for 8 and 5 days at 4 and 10°C respectively in yogurt at pH 4.17, 17 and 15 days at 4 and 10°C respectively in yogurt at pH 4.39, and 17days at both 4 and 10°C in yogurt at pH 4.47. E. coli O157:H7 did not survive cooking procedures at 56°C in Cottage cheese. However, the pathogen survived for 27, 30, and 27 days in Colby, Romano, and Feta cheeses respectively. A high correlation of r2 > 0.89 was obtained between counts of bioluminescenct colonies and standard plate count for all yogurt and cheese varieties, indicating that bioluminescence was a sensitive and rapid indicator of cellular viability for E. coli O157:H7. Survival of the pathogen, as indicated by this method, is possible in highly acidic environments even at refrigeration temperatures. This poses a potential hazard should postprocessing contamination occur.

Recombinant bacteriophages specific for Salmonella spp. and containing bacterial luciferase genes were constructed. The phage caused the host cells to luminesce when mixed with Salmonella spp. and the luminescence could be detected using a photon-counting charge-coupled device (CCD) camera, a luminometer, or X-ray film. The initial assay system was capable of detecting Salmonella isolates from group B and group D. Certain isolates from group C could also be detected. With 6 h of preincubation, as few as 10 CFU of Salmonella cells per ml in the original sample could be detected. The minimum time required for the detection of 10

The meat industry is in need of faster and more reliable methods to determine microbial loads in food products. A rapid method (

An investigation was conducted to assess the practical use of an adenosine triphosphate (ATP) bioluminescence assay to evaluate the effectiveness of cleaning and sanitizing meat slicers in eight health care institutions. The ATP bioluminescence assay was compared to conventional swabbing techniques using standard plate count to enumerate microbial load. Assays were performed on meat slicers before use, after slicing a meat product and after sanitizing. There was a general overall agreement in results obtained by both methods but the ATP assay gave a better indication of the cleanliness of the meat slicer as it was able to detect the presence of meat residues left on the blade after improper sanitation. Results were available within 5 min using the ATP bioluminescence method, thus providing an opportunity for immediate remedial action.

Psychrotrophic bacteria have been recognized as a recurring problem in the refrigerated storage and distribution of fluid milk and cream and other perishable dairy products for several decades. Much emphasis has been focused on postpasteurization contaminants that are psychrotrophic, (e.g., Pseudomonas , Flavobacterium , and Alcaligenes spp.). Common sources of these gram-negative, non-sporeforming organisms are equipment surfaces and water supplies. Although these organisms are generally heat sensitive, many of their associated proteinases and lipases can withstand moderate to severe heat treatments and cause product deterioration. With the advance of improved control of postpasteurization contamination by nonheat-resistant psychrotrophs, more recent attention has been directed at psychrotrophic sporeformers and their potential impact on milk quality and shelf life properties. Heat-resistant psychrotrophs include members from the genera Clostridium , Arthrobacter , Microbacterium , Streptococcus , Corynebacterium , and Bacillus . However, the predominant microorganisms which comprise this category are Bacillus species. These bacteria can be introduced into milk supplies from water, udder and teat surfaces, or from soil and milkstone deposits on farm bulk tanks, pumps, pipelines, gaskets, and processing equipment. There is speculation that they can also be postpasteurization contaminants. When in the spore state, these microorganisms easily survive the typical range of pasteurization conditions with subsequent germination and outgrowth of vegetative cells. These organisms produce degradative enzymes (e.g., proteinases, lipases, and phospholipases) similar to those of non-sporeforming psychrotrophs. Enzymatic activity results in the development of objectionable flavor and quality defects in dairy products. The unique combination of both heat-resistant and psychrotrophic properties with the same microorganism represents substantial potential for causing spoilage of perishable milk products. Recent trends of higher pasteurization temperatures and extended refrigerated storage time of both raw and pasteurized milk and cream products exacerbates the significance of this group of microorganisms for the dairy foods industry.

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.

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.

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.

The surface microbiota of washed-rind cheeses is variable and complex. The processes involved in the production of these cheeses are not fully understood and pathogenic bacteria have been occasionally reported in the smear on the surface of the cheeses. This review describes the prevalent factors that determine development of the complex consortia of microorganisms in the smear of washed-rind cheeses, with a focus on factors that encourage or suppress growth of Listeria monocytogenes . The natural microflora in smears have been shown to exert antilisterial activities (e.g., bacteriocins), which can be utilized to develop commercial starter and smear cultures. The potential of bacteriophage to control pathogens is also being researched. A more detailed knowledge of the microbial ecology of washed-rind cheeses will reveal opportunities to maintain and improve cheese safety and quality.

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 (

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.

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 (Fe

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.

Based on morphology and comparative nucleotide and protein sequence analysis, a new subfamily of the family Siphoviridae is proposed, named "Jerseyvirinae" and consisting of three genera, "Jerseylikevirus", "Sp3unalikevirus" and "K1glikevirus". To date, this subfamily consists of 18 phages for which the genomes have been sequenced. Salmonella phages Jersey, vB_SenS_AG11, vB_SenS-Ent1, vB_SenS-Ent2, vB_SenS-Ent3, FSL SP-101, SETP3, SETP7, SETP13, SE2, SS3e and wksl3 form the proposed genus "Jerseylikevirus". The proposed genus "K1glikevirus" consists of Escherichia phages K1G, K1H, K1ind1, K1ind2 and K1ind3. The proposed genus "Sp3unalikevirus" contains one member so far. Jersey-like phages appear to be widely distributed, as the above phages were isolated in the UK, Canada, the USA and South Korea between 1970 and the present day. The distinguishing features of this subfamily include a distinct siphovirus morphotype, genomes of 40.7-43.6 kb (49.6-51.4 mol % G +C), a syntenic genome organisation, and a high degree of nucleotide sequence identity and shared proteins. All known members of the proposed subfamily are strictly lytic.

Virulent bacteriophages (VP) and high hydrostatic pressure (HHP) were studied for the inactivation of Shigella flexneri in ground beef and Vibrio cholerae in salmon and mussels. Inoculated foods were treated individually with HHP (150-450 MPa for 5 and 9 min, 300 MPa for 13 min, and 550 MPa for 5 min), with phages (cocktail of 3 S. flexneri or single V. cholerae phages, both applied at 109 PFU/mL) or combinations thereof (HHP/VP, VP/HHP). Stand-alone treatments with VP, HHP below 450 MPa (seafood) or 550 MPa (meat), and combined treatments of VP and HHP at 250 MPa for 5 min did not reduce bacterial counts below the detection limit. By contrast, complete inactivation of S. flexneri and V. cholerae (P

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.

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.

Wild-type T4 bacteriophage and recombinant reporter lac Z T4 bacteriophage carrying the β-galactosidase gene were used for detection of generic Escherichia coli by monitoring the release of β-galactosidase upon phage-mediated cell lysis. The reaction was performed on a paper-based portable culture device to limit the diffusion of reagents and, hence, increase the sensitivity of the assay, and to avoid handling large sample volumes, making the assay suitable for on-site analysis. Chromogenic (chlorophenol red-β-d-galactopyranoside, CPRG) and bioluminescent (6-O-β-galactopyranosyl-luciferin, Beta-Glo®) β-galactosidase substrates were tested in the assay. Water samples were first filtered through 0.45-μm pore size filters to concentrate bacteria. The filters were then placed into the paper-based device containing nutrient medium and incubated at 37 °C for 4 h. Bacteriophage with the respective indicator substrate was added to the device, and signal (color, luminescence) development was recorded with a digital camera, luminometer, or luminescence imaging device. It was demonstrated that as low as 40 or

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne’s disease, has a doubling time of 24 hours, making rapid detection very difficult. Mycobacteriophages can be used in the detection of disease-causing mycobacteria such as MAP. Isolation and sequencing the genomes of lytic MAP bacteriophages are important preliminary steps towards designing phage-based rapid detection assays for this bacterium. A simple optimized protocol was developed to allow reproducible production of confluent growth of MAP on plates within four to six weeks of incubation at 30 °C. This protocol was applied to the screening of environmental and fecal samples for bacteriophages inhibiting the growth of MAP. As a result, a lytic phage, vB_MapS_FF47, was isolated from bovine feces. FF47 contains a double-stranded DNA genome ~48 kb in length with 73 protein coding sequences. It does not carry temperate or known virulence genes. This phage was shown to be most closely related to Mycobacterium phage Muddy, isolated in South Africa, and Gordonia phage GTE2; however, it could not infect any of the tested Gordonia, Rhodococcus, or Nocardia spp. that GTE2 could. The protocols that were developed for growth and phage isolation have potential applications in a high-throughput screening for compounds inhibiting the growth of MAP. This work describes the first time that a phage was isolated against M. paratuberculosis.

Phage-based detection assays have been developed for the detection of viable bacteria for applications in clinical diagnosis, monitoring of water quality, and food safety. The majority of these assays deliver a positive readout in the form of newly generated progeny phages by the bacterial host of interest. Progeny phages are often visualized as plaques, or holes, in a lawn of bacteria on an agar-filled Petri dish; however, this rate-limiting step requires up to 12 h of incubation time. We have previously described an amplification of bacteriophages M13 inside droplets of media suspended in perfluorinated oil; a single phage M13 in a droplet yields 10(7) copies in 3-4 h. Here, we describe that encapsulation of reporter phages, both lytic T4-LacZ and nonlytic M13, in monodisperse droplets can also be used for rapid enumeration of phage. Compartmentalization in droplets accelerated the development of the signal from the reporter enzyme; counting of "positive" droplets yields accurate enumeration of phage particles ranging from 10(2) to 10(6) pfu/mL. For enumeration of T4-LacZ phage, the fluorescent signal appeared in as little as 90 min. Unlike bulk assays, quantification in emulsion is robust and insensitive to fluctuations in environmental conditions (e.g., temperature). Power-free emulsification using gravity-driven flow in the absence of syringe pumps and portable fluorescence imaging solutions makes this technology promising for use at the point of care in low-resource environments. This droplet-based phage enumeration method could accelerate and simplify point-of-care detection of the pathogens for which reporter bacteriophages have been developed.

F-specific (F+) RNA phages are recommended as indicators of fecal contamination and the presence of enteric viruses and as viral surrogates to elucidate the resistance of viruses to adverse conditions or to assess the effectiveness of inactivating processes. Reverse transcription (RT)-PCR methods have been used to detect, quantify, or identify subgroups of F+ RNA phages. However, these methods may overestimate the infectivity of F+ RNA phages in test samples, since the presence of both infectious and inactivated phages (or naked RNA) can lead to positive RT-PCR signals. In this study, we evaluated the ability of an enzyme treatment (ET) with proteinase K and RNase A prior to RNA extraction, followed by RT-PCR, to differentiate infectious and inactivated F+ RNA phages. The results indicated that ET RT-PCR reduced, but did not completely eliminate, false-positive signals encountered with RT-PCR alone. The two-step ET RT-PCR, in which the enzymes were added sequentially, was more effective at reducing false-positive signals than the one-step ET RT-PCR, which involved addition of both enzymes together. Despite its inability to completely eliminate false-positive signals, ET RT-PCR gave more reliable information on the infectivity of F+ RNA phages. Thus, the method is better than RT-PCR alone for detecting F+ RNA phages as indicators to assess the risk of fecal contamination by enteric pathogens or to evaluate the effectiveness of virus-inactivating processes.

The milk fat globule membrane (MFGM), the material surrounding milk fat globules, is not only interesting from a technological standpoint but it also shows great potential as a health ingredient, as it exerts cytotoxic and apoptotic effects against colon cancer cells. Although the effects of milk processing on the MFGM composition and functionality are well documented, less is understood on how processing may affect its bioactivity. This study aimed to determine if heating can affect the antiproliferative capacity of the MFGM. To do so, MFGM was extracted from milk heated at 80 °C for 10 min, as this temperature/time regime is known to cause extensive protein-protein interactions with changes in the processing functionality of milk. Two cell lines, whose morphological features are representative of two different stages of colon carcinogenesis (HT-29 and Caco-2), were used to test the antiproliferative capacity of MFGM isolates obtained either from untreated or heated milk. Cell proliferation analysis showed a similar dose-dependent decrease of DNA synthesis in both cell lines exposed to 6.25–200 μg of MFGM protein.mL−1, isolated from unheated milk. The heat treatment diminished the efficacy of the MFGM isolates, as only the highest concentrations of MFGM tested following heating showed a significant effect on cell proliferation. The decreased ability of MFGM isolates to affect the carcinoma cell proliferation was attributed to changes in composition, mainly phospholipid losses. Changes to the supramolecular structure of the MFGM caused by heating may also have played a role. This work demonstrates the importance of processing history in assessing the biological functionality of MFGM.

Cronobacter sakazakii, an opportunistic pathogen found in milk-based powdered infant formulae, has been linked to meningitis in infants, with high fatality rates. A set of phages from various environments were purified and tested in vitro against strains of C. sakazakii. Based on host range and lytic activity, the T4-like phage vB_CsaM_GAP161, which belongs to the family Myoviridae, was selected for evaluation of its efficacy against C. sakazakii. Galleria mellonella larvae were used as a whole-animal model for pre-clinical testing of phage efficiency. Twenty-one Cronobacter strains were evaluated for lethality in G. mellonella larvae. Different strains of C. sakazakii caused 0 to 98 % mortality. C. sakazakii 3253, with an LD50 dose of ~2.0 × 105 CFU/larva (24 h, 37 °C) was selected for this study. Larvae infected with a dose of 5 × LD50 were treated with phage GAP161 (MOI = 8) at various time intervals. The mortality rates were as high as 100 % in the groups injected with bacteria only, compared to 16.6 % in the group infected with bacteria and treated with phage. Phage GAP161 showed the best protective activity against C. sakazakii when the larvae were treated prior to or immediately after infection. The results obtained with heat-inactivated phage proved that the survival of the larvae is not due to host immune stimulation. These results suggest that phage GAP161 is potentially a useful control agent against C. sakazakii. In addition, G. mellonella may be a useful whole-animal model for pre-screening phages for efficacy and safety prior to clinical evaluation in mammalian models.

The nutritional value and characterization of minor milk components on mammalian immune function are not fully understood. The aim of this research was to test the ability of a milk fat globule membrane (MFGM) isolate to modulate murine immune function in vitro, by studying its effects on splenocyte proliferation, apoptosis, and cytokine production. Proliferation of spleen cells was not affected by the MFGM isolate; however, in the presence of polyclonal activators, the MFGM isolate suppressed cell proliferation. Results obtained by flow cytometry did not support programmed cell death as the cause of the MFGM immune-modulating capacity. A mode of suppression on the splenocyte activation process was suggested from a marked decrease in the production of IFN-γ and tumor necrosis factor-α cytokines, typical indicators of immune cell activation. The effect of MFGM on IL-4 secretion was significantly less than that for the other 2 cytokines. The activity exerted by the MFGM over concanavalin A-stimulated cells differed from that observed in cells treated with lipopolysaccharide, suggesting a different mode of action depending on the activator used. These results indicate the potential of MFGM extracts as functional ingredients with bioactive modulating capacity.

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.

Campylobacter spp. are among the most commonly reported bacterial causes of acute diarrheal disease in humans worldwide. Potential virulence factors include motility, chemotaxis, colonization ability, adhesion to intestinal cells, invasion and epithelial translocation, intracellular survival, and formation of toxins. Probiotic Lactobacillus and Bifidobacterium strains are known to have an inhibitory effect against the growth of various foodborne pathogens. The objective of this study was to investigate the effect of Lactobacillus acidophilus strain La-5 and Bifidobacterium longum strain NCC2705 cell-free spent media (CFSM) on the expression of virulence genes (cadF, cdtB, flaA, and ciaB) of Campylobacter jejuni strain 81-176 and a luxS mutant, using real-time PCR. Our results demonstrated that the CFSM of both probiotic strains were able to down-regulate the expression of ciaB (ratio of -2.80 and -5.51, respectively) and flaA (ratio of -7.00 and -5.13, respectively) in the wild-type Campylobacter strain. In the luxS mutant, where the activated methyl cycle is disrupted, only the ciaB gene (ratio -7.21) was repressed in the presence of La-5 CFSM. A supplementation of homocysteine to restore the disrupted cycle was able to partially reestablish the probiotic effect of both strains. luxS and the activated methyl cycle might play an active role in the modulation of virulence of C. jejuni by probiotic extracts.