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The growing human population is currently facing an unprecedented challenge on global food production and sustainability. Despite recognizing poultry as one of the most successful and rapidly growing food industries to address this challenge; poultry health and safety remain major issues that entail immediate attention. Bacterial diseases including colibacillosis, salmonellosis, and necrotic enteritis have become increasingly prevalent during poultry production. Likewise, outbreaks caused by consumption of undercooked poultry products contaminated with zoonotic bacterial pathogens such as Salmonella, Campylobacter and Listeria, are a serious public health concern. With antimicrobial resistance problem and restricted use of antibiotics in food producing animals, bacteriophages are increasingly recognized as an attractive natural antibacterial alternative. Bacteriophages have recently shown promising results to treat diseases in poultry, reduce contamination of carcasses, and enhance the safety of poultry products. Omics technologies have been successfully employed to accurately characterize bacteriophages and their genes/proteins important for interaction with bacterial hosts. In this review, the potential of using lytic bacteriophages to mitigate the risk of major poultry-associated bacterial pathogens are explored. This study also explores challenges associated with the adoption of this technology by industries. Furthermore, the impact of omics approaches on studying bacteriophages, their host interaction and applications is discussed.

Here, we report the genome sequence of a jumbo Escherichia phage vB_EcoM_EC001, a myovirus isolated from primary sludge using enterohemorrhagic Escherichia coli O157:H7. The genome is 240,200 bp long and has 270 predicted coding sequences, including a tryptophanyl tRNA gene. It belongs to genus Seoulvirus .

Bacteriophages are viruses that infect bacteria and are present in niches where bacteria thrive. In recent years, the suggested application areas of lytic bacteriophage have been expanded to include therapy, biocontrol, detection, sanitation, and remediation. However, phage application is constrained by the phage’s host range—the range of bacterial hosts sensitive to the phage and the degree of infection. Even though phage isolation and enrichment techniques are straightforward protocols, the correlation between the enrichment technique and host range profile has not been evaluated. Agar-based methods such as spotting assay and efficiency of plaquing (EOP) are the most used methods to determine the phage host range. These methods, aside from being labor intensive, can lead to subjective and incomplete results as they rely on qualitative observations of the lysis/plaques, do not reflect the lytic activity in liquid culture, and can overestimate the host range. In this study, phages against three bacterial genera were isolated using three different enrichment methods. Host range profiles of the isolated phages were quantitatively determined using a high throughput turbidimetric protocol and the data were analyzed with an accessible analytic tool “PHIDA”. Using this tool, the host ranges of 9 Listeria, 14 Salmonella, and 20 Pseudomonas phages isolated with different enrichment methods were quantitatively compared. A high variability in the host range index (HRi) ranging from 0.86–0.63, 0.07–0.24, and 0.00–0.67 for Listeria, Salmonella, and Pseudomonas phages, respectively, was observed. Overall, no direct correlation was found between the phage host range breadth and the enrichment method in any of the three target bacterial genera. The high throughput method and analytics tool developed in this study can be easily adapted to any phage study and can provide a consensus for phage host range determination.

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.

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.

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.

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.

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.

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

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.

The ability of phages to specifically interact with and lyse their host bacteria makes them ideal antibacterial agents. The range of applications of bacteriophage can be extended by their immobilization on inert surfaces. A novel method for the oriented immobilization of bacteriophage has been developed. The method was based on charge differences between the bacteriophage head, which exhibits an overall net negative charge, and the tail fibers, which possess an overall net positive charge. Hence, the head would be more likely to attach to positively charged surfaces, leaving the tails free to capture and lyse bacteria. Cellulose membranes modified so that they had a positive surface charge were used as the support for phage immobilization. It was established that the number of infective phages immobilized on the positively charged cellulose membranes was significantly higher than that on unmodified membranes. Cocktails of phages active against Listeria or Escherichia coli immobilized on these membranes were shown to effectively control the growth of L. monocytogenes and E. coli O157:H7 in ready-to-eat and raw meat, respectively, under different storage temperatures and packaging conditions. The phage storage stability was investigated to further extend their industrial applications. It was shown that lyophilization can be used as a phage-drying method to maintain their infectivity on the newly developed bioactive materials. In conclusion, utilizing the charge difference between phage heads and tails provided a simple technique for oriented immobilization applicable to a wide range of phages and allowed the retention of infectivity.

Bacteriophages are selective anti-bacterial agents, which are receiving increasing acceptance by regulatory agencies for use both in the food industry and in clinical settings for biocontrol. While immobilized phage could be particularly useful to create antimicrobial surfaces, current immobilization strategies require chemical bioconjugation to surfaces or more difficult processes involving modification of their head proteins to express specific binding moieties, for example, biotin or cellulose binding domains; procedures that are both time and money intensive. We report that morphologically different bacteriophages, active against a variety of food-borne bacteria: Escherichia coli; Salmonella enterica; Listeria monocytogenes; and Shigella boydii, will effectively physisorb to silica particles, prepared by silica surface modification with poly(ethylene glycol), carboxylic acid groups, or amines. The phages remain infective to their host bacteria while adsorbed on the surface of the silica particles. The number of infective phage bound to the silica is enhanced by the presence of ionic surfaces, with greater surface charge - to a maximum - correlating with greater concentration of adsorbed phage. Above the maximum charge concentration, the number of active phage drops.

Even though high hygiene and sanitation standards are promoted in the food industry, pathogens are not always eradicated from the finished product or from the food-processing environment. Thus, there is always interest in the development of additional ways to improve the safety of food. The idea of destroying pathogens by natural interventions is very attractive. In this context, bacteriophages (phages) have emerged as a new biocontrol tool, which presents enormous potential in the fight to reduce the burden of infectious diseases. In this chapter we will review phage history and biology, and the methods that have been proposed to use phages to control bacterial pathogens in food.

This study aimed to isolate and characterize bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces. Of 37 non-O157 STEC-infecting phages isolated, those targeting O26 (AXO26A, AYO26A, AYO26B), O103 (AXO103A, AYO103A), O111 (AXO111A, AYO111A), O121 (AXO121A, AXO121B), and O145 (AYO145A, AYO145B) were further characterized. Transmission electron microscopy showed that the 11 isolates belonged to 3 families and 6 genera: the families Myoviridae (types rV5, T4, ViI, O1), Siphoviridae (type T5), and Podoviridae (type T7). Genome size of the phages as determined by pulsed-field gel electrophoresis ranged from 38 to 177 kb. Excluding phages AXO26A, AYO103A, AYO145A, and AYO145B, all other phages were capable of lysing more than 1 clinically important strain from serogroups of O26, O91, O103, O111, O113, O121, and O128, but none exhibited infectivity across all serogroups. Moreover, phages AYO26A, AXO121A, and AXO121B were also able to lyse 4 common phage types of STEC O157:H7. Our findings show that a diversity of non-O157 STEC-infecting phages are harbored in bovine feces. Phages AYO26A, AYO26B, AXO103A, AXO111A, AYO111A, AXO121A, and AXO121B exhibited a broad host range against a number of serogroups of STEC and have potential for the biocontrol of STEC in the environment.

Aims To produce light-activated antimicrobial materials composed of the photodynamic dye phloxine B incorporated into paper or cellulose membranes and to investigate ability of these materials to decrease bacterial loads on their surfaces as well as on food surfaces that were in contact with these materials under illumination with regular white light. Methods and Results Antimicrobial cellulose-based materials with incorporated phloxine B were produced using a layer-by-layer deposition method. Antimicrobial properties of the materials were tested in model systems as well as for decontamination of food and food contact surfaces. Pseudomonas aeruginosa, Listeria monocytogenes and Bacillus anthracis were efficiently killed by exposure of the bacterial suspension to the dye-containing material under illumination with white light, but Salmonella Typhimurium and Escherichia coli O157:H7 were only partially affected. Application of the materials for decontamination of food surfaces artificially contaminated with L. monocytogenes was shown to be ineffective, while the self-decontamination of the material surface by exposure to white light resulted in eradication of L. monocytogenes cells from the material surface. Conclusions The developed materials showed significant self-decontaminating ability when under illumination; however, decontamination of food surfaces in contact with the developed materials was not achieved. Significance and Impact of the Study The study demonstrates the antibacterial activity of materials with incorporated photodynamic dyes when under illumination with regular-intensity white light. Possible uses of the light-activated antimicrobial materials for food processing, as food contact surfaces, and surfaces in public areas to prevent cross-contamination are discussed.

Pulsed electric fields (PEF), heat-assisted PEF (H-PEF), and virulent bacteriophage (VP) are non-thermal techniques for pathogen inactivation in liquids that were investigated individually, and in combination (PEF/VP, H-PEF/VP) to control enterohemorrhagic Escherichia coli (EHEC) O157:H7 in Luria-Bertani broth (LBB) and Ringer's solution (RS). Treated cells were subsequently incubated at refrigeration (4°C) and temperature-abuse conditions (12°C) for 5 days. When EHEC cells grown in LBB were subjected to non-thermal processing and subsequently stored at 12°C for 5 days, reductions in count of between 0.1 and 0.6 log cycles were observed and following storage at 4°C the decrease in counts varied between 0.2 and 1.1 log10. For bacteria cells suspended in RS values ranged from 0.1 to ≥3.9 log cycles at both storage temperatures. The most effective treatments were H-PEF and H-PEF/VP, both producing a >3.4 log cycle reduction of cells suspended in non-nutrient RS. Analysis of EHEC recovery on selective and non-selective media indicated no occurrence of sub-lethal damage for VP, PEF/VP, and H-PEF/VP-treated cells. The findings indicate that combining PEF and lytic phage may represent a suitable alternative to conventional fluid decontamination following further process optimization. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:110–118, 2015

This chapter presents recent advances in bacteriophage research and their application in the area of food safety. Section 1 describes general facts on phage biology that are relevant to their application for control and detection of bacterial pathogens in food and environmental samples. Section 2 summarizes the recently acquired data on application of bacteriophages to control growth of bacterial pathogens and spoilage organisms in food and food-processing environment. Section 3 deals with application of bacteriophages for detection and identification of bacterial pathogens. Advantages of bacteriophage-based methods are presented and their shortcomings are discussed. The chapter is intended for food scientist and food product developers, and people in food inspection and health agencies with the ultimate goal to attract their attention to the new developing technology that has a tremendous potential in providing means for producing wholesome and safe food.

We suggest a bacteriophage genus, “Viunalikevirus”, as a new genus within the family Myoviridae. To date, this genus includes seven sequenced members: Salmonella phages ViI, SFP10 and ΦSH19; Escherichia phages CBA120 and PhaxI; Shigella phage phiSboM-AG3; and Dickeya phage LIMEstone1. Their shared myovirus morphology, with comparable head sizes and tail dimensions, and genome organization are considered distinguishing features. They appear to have conserved regulatory sequences, a horizontally acquired tRNA set and the probable substitution of an alternate base for thymine in the DNA. A close examination of the tail spike region in the DNA revealed four distinct tail spike proteins, an arrangement which might lead to the umbrella-like structures of the tails visible on electron micrographs. These properties set the suggested genus apart from the recently ratified subfamily Tevenvirinae, although a significant evolutionary relationship can be observed.

This chapter presents recent advances in bacteriophage research and their application in the area of food safety. Section 1 describes general facts on phage biology that are relevant to their application for control and detection of bacterial pathogens in food and environmental samples. Section 2 summarizes the recently acquired data on application of bacteriophages to control growth of bacterial pathogens and spoilage organisms in food and food-processing environment. Section 3 deals with application of bacteriophages for detection and identification of bacterial pathogens. Advantages of bacteriophage-based methods are presented and their shortcomings are discussed. The chapter is intended for food scientist and food product developers, and people in food inspection and health agencies with the ultimate goal to attract their attention to the new developing technology that has a tremendous potential in providing means for producing wholesome and safe food.