A Bacteriophage Cocktail Targeting Yersinia pestis Provides Strong Post-Exposure Protection in a Rat Pneumonic Plague Model.

Yersinia pestis , one of the deadliest bacterial pathogens ever known, is responsible for three plague pandemics and several epidemics, with over 200 million deaths during recorded history. Due to high genomic plasticity, Y. pestis is amenable to genetic mutations as well as genetic engineering that can lead to the emergence or intentional development of pan-drug resistant strains. The dissemination of such Y. pestis strains could be catastrophic, with public health consequences far more daunting than those caused by the recent COVID-19 pandemic. Thus, there is an urgent need to develop novel, safe, and effective treatment approaches for managing Y. pestis infections. This includes infections by antigenically distinct strains for which vaccines, none FDA approved yet, may not be effective, and those that cannot be controlled by approved antibiotics. Lytic bacteriophages provide one such alternative approach. In this study, we examined post-exposure efficacy of a bacteriophage cocktail, YPP-401, to combat pneumonic plague caused by Y. pestis CO92. YPP-401 is a four-phage preparation with a 100% lytic activity against a panel of 68 genetically diverse Y. pestis strains. Using a pneumonic plague aerosol challenge model in gender-balanced Brown Norway rats, YPP-401 demonstrated ∼88% protection when delivered 18 hours post-exposure for each of two administration routes (i.e., intraperitoneal and intranasal) in a dose-dependent manner. Our studies suggest that YPP-401 could provide an innovative, safe, and effective approach for managing Y. pestis infections, including those caused by naturally occurring or intentionally developed strains that cannot be managed by vaccines in development and antibiotics.

ECOPHAGE: Combating Antimicrobial Resistance Using Bacteriophages for Eco-Sustainable Agriculture and Food Systems.

The focus of this meeting was to discuss the suitability of using bacteriophages as alternative antimicrobials in the agrifood sector. Following a One Health approach, the workshop explored the possibilities of implementing phage application strategies in the agriculture, animal husbandry, aquaculture, and food production sectors. Therefore, the meeting had gathered phage researchers, representatives of the agrifood industry, and policymakers to debate the advantages and potential shortcomings of using bacteriophages as alternatives to traditional antimicrobials and chemical pesticides. Industry delegates showed the latest objectives and demands from consumers. Representatives of regulatory agencies (European Medicines Agency (EMA) and Spanish Agency of Medicines and Health Products (AEMPS)) presented an update of new regulatory aspects that will impact and support the approval and implementation of phage application strategies across the different sectors.

Phage biocontrol for reducing bacterial foodborne pathogens in produce and other foods.

Foodborne pathogen contamination causes approximately 47 million cases of foodborne illness in the United States and renders thousands of pounds of food products inedible, aggravating the already dire situation of food loss. Reducing foodborne contamination not only improves overall global public health but also reduces food waste and loss. Phage biocontrol or phage-mediated reduction of bacterial foodborne pathogens in various foods has been gaining interest recently as an effective and environmentally friendly food-safety approach. Consequently, several commercial phage-based food-safety products have been developed and are increasingly implemented by the food industry in the United States. This review focuses on the use of phage biocontrol in mitigating bacterial pathogen contamination in various food products with a special emphasis on applications to fresh produce.

Safety and Efficacy of an AIEC-targeted Bacteriophage Cocktail in a Mice Colitis Model.

BACKGROUND AND AIMS: Adherent invasive Escherichia coli [AIEC] are recovered with a high frequency from the gut mucosa of Crohn's disease patients and are believed to contribute to the dysbiosis and pathogenesis of this inflammatory bowel disease. In this context, bacteriophage therapy has been proposed for specifically targeting AIEC in the human gut with no deleterious impact on the commensal microbiota. METHODS: The in vitro efficacy and specificity of a seven lytic phage cocktail [EcoActive™] was assessed against [i] 210 clinical AIEC strains, and [ii] 43 non-E. coli strains belonging to the top 12 most common bacterial genera typically associated with a healthy human microbiome. These data were supported by in vivo safety and efficacy assays conducted on healthy and AIEC-colonized mice, respectively. RESULTS: The EcoActive cocktail was effective in vitro against 95% of the AIEC strains and did not lyse any of the 43 non-E. coli commensal strains, in contrast to conventional antibiotics. Long-term administration of the EcoActive cocktail to healthy mice was safe and did not induce dysbiosis according to metagenomic data. Using a murine model of induced colitis of animals infected with the AIEC strain LF82, we found that a single administration of the cocktail failed to alleviate inflammatory symptoms, while mice receiving the cocktail twice a day for 15 days were protected from clinical and microscopical manifestations of inflammation. CONCLUSIONS: Collectively, the data support the approach of AIEC-targeted phage therapy as safe and effective treatment for reducing AIEC levels in the gut of IBD patients.

A Bacteriophage Cocktail Significantly Reduces Listeria monocytogenes without Deleterious Impact on the Commensal Gut Microbiota under Simulated Gastrointestinal Conditions.

In this study, we examined the effect of a bacteriophage cocktail (tentatively designated as the Foodborne Outbreak Pill (FOP)) on the levels of Listeria monocytogenes in simulated small intestine, large intestine, and Caco-2 model systems. We found that FOP survival during simulated passage of the upper gastrointestinal was dependent on stomach pH, and that FOP robustly inhibited L. monocytogenes levels with effectiveness comparable to antibiotic treatment (ampicillin) under simulated ilium and colon conditions. The FOP did not inhibit the commensal bacteria, whereas ampicillin treatment led to dysbiosis-like conditions. The FOP was also more effective than an antibiotic in protecting Caco-2 cells from adhesion and invasion by L. monocytogenes (5-log reduction vs. 1-log reduction) while not triggering an inflammatory response. Our data suggested that the FOP may provide a robust protection against L. monocytogenes should the bacterium enter the human gastrointestinal tract (e.g., by consumption of contaminated food), without deleterious impact on the commensal bacteria.

Prophylactic Administration of a Bacteriophage Cocktail Is Safe and Effective in Reducing Salmonella enterica Serovar Typhimurium Burden in Vivo.

Nontyphoidal Salmonella bacteria are the causative agent of salmonellosis, which accounts for the majority of foodborne illness of bacterial etiology in humans. Here, we demonstrate the safety and efficacy of the prophylactic administration of a bacteriophage preparation termed FOP (foodborne outbreak pill), which contains lytic phages targeting Salmonella (SalmoFresh phage cocktail), Shiga toxin-producing Escherichia coli (STEC), and Listeria monocytogenes, for lowering Salmonella burdens in OMM12 gnotobiotic mice. Prophylactic administration of FOP significantly reduced the levels of Salmonella in feces and in intestinal sections compared to the levels in controls. Moreover, the overall symptoms of the disease were also considerably lessened. Dose-dependent administration of FOP showed that phage amplification reached similarly high levels in less than 48 h independent of dose. In addition, 16S rRNA gene analysis showed that FOP did not alter the intestinal microbiota of healthy OMM12 mice and reduced microbiota perturbations induced by Salmonella. FOP maintained its full potency against Salmonella in comparison to that of SalmoFresh, its Salmonella-targeting component phages alone. Altogether, the data support that preventive administration of FOP may offer a safe and effective approach for reducing the risk of foodborne infections caused by Salmonella and, potentially, other foodborne bacteria (namely, STEC and L. monocytogenes) targeted by the FOP preparation. IMPORTANCE Foodborne bacterial infections cause worldwide economic loss. During an epidemic, the use of antibiotics to slow down the spread of the disease is not recommended because of their side effects on the resident microbiota and the selection of antibiotic-resistant bacteria. Here, we investigated the potential for the prophylactic administration of bacteriophages (viruses infecting bacteria) to reduce the burden of Salmonella in vivo using mice colonized by a synthetic microbiota. We found that the repeated administration of bacteriophages was safe and efficient in lowering the Salmonella burden. Perturbations of the microbiota by the Salmonella infection were also reduced when mice received bacteriophages. Altogether, these data support the use of bacteriophages as a prophylactic intervention to lower the spread of foodborne epidemics.

Bacteriophages against Vibrio coralliilyticus and Vibrio tubiashii: Isolation, Characterization, and Remediation of Larval Oyster Mortalities.

Vibrio coralliilyticus and Vibrio tubiashii are pathogens responsible for high larval oyster mortality rates in shellfish hatcheries. Bacteriophage therapy was evaluated to determine its potential to remediate these mortalities. Sixteen phages against V. coralliilyticus and V. tubiashii were isolated and characterized from Hawaiian seawater. Fourteen isolates were members of the Myoviridae family, and two were members of the Siphoviridae In proof-of-principle trials, a cocktail of five phages reduced mortalities of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) by up to 91% 6 days after challenge with lethal doses of V. coralliilyticus Larval survival depended on the oyster species, the quantities of phages and vibrios applied, and the species and strain of Vibrio A later-generation cocktail, designated VCP300, was formulated with three lytic phages subsequently named Vibrio phages vB_VcorM-GR7B, vB_VcorM-GR11A, and vB_VcorM-GR28A (abbreviated 7B, 11A, and 28A, respectively). Together, these three phages displayed host specificity toward eight V. coralliilyticus strains and a V. tubiashii strain. Larval C. gigas mortalities from V. coralliilyticus strains RE98 and OCN008 were significantly reduced by >90% (P < 0.0001) over 6 days with phage treatment compared to those of untreated controls. Genomic sequencing of phages 7B, 11A, and 28A revealed 207,758-, 194,800-, and 154,046-bp linear DNA genomes, respectively, with the latter showing 92% similarity to V. coralliilyticus phage YC, a strain from the Great Barrier Reef, Australia. Phage 7B and 11A genomes showed little similarity to phages in the NCBI database. This study demonstrates the promising potential for phage therapy to reduce larval oyster mortalities in oyster hatcheries.IMPORTANCE Shellfish hatcheries encounter episodic outbreaks of larval oyster mortalities, jeopardizing the economic stability of hatcheries and the commercial shellfish industry. Shellfish pathogens like Vibrio coralliilyticus and Vibrio tubiashii have been recognized as major contributors of larval oyster mortalities in U.S. East and West Coast hatcheries for many years. This study isolated, identified, and characterized bacteriophages against these Vibrio species and demonstrated their ability to reduce mortalities from V. coralliilyticus in larval Pacific oysters and from both V. coralliilyticus and V. tubiashii in larval Eastern oysters. Phage therapy offers a promising approach for stimulating hatchery production to ensure the well-being of hatcheries and the commercial oyster trade.

Phage Biocontrol Applications in Food Production and Processing.

Bacteriophages, or phages, are one of the most, if not the most, ubiquitous organisms on Earth. Interest in various practical applications of bacteriophages has been gaining momentum recently, with perhaps the most attention (and most regulatory approvals) focused on their use to improve food safety. This approach, termed 'phage biocontrol' or 'bacteriophage biocontrol', includes both pre- and post-harvest application of phages as well as decontamination of the food contact surfaces in food processing facilities. This review focuses on post-harvest applications of phage biocontrol, currently the most commonly used type of phage mediation. We also briefly describe various commercially available phage preparations and discuss the challenges still facing this novel yet promising approach.

Phage Biocontrol Improves Food Safety by Significantly Reducing the Level and Prevalence of Escherichia coli O157:H7 in Various Foods.

ABSTRACT: Management of Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, in food products is a major challenge for the food industry. Several interventions, such as irradiation, chemical disinfection, and pasteurization, have had variable success controlling STEC contamination. However, these interventions also indiscriminately kill beneficial bacteria in foods, may impact organoleptic properties of foods, and are not always environmentally friendly. Biocontrol using bacteriophage-based products to reduce or eliminate specific foodborne pathogens in food products has been gaining attention due to the specificity, safety, and environmentally friendly properties of lytic bacteriophages. We developed EcoShield PX, a cocktail of lytic bacteriophages, that specifically targets STEC. This study was conducted to examine the efficacy of this bacteriophage cocktail for reducing the levels of E. coli O157:H7 in eight food products: beef chuck roast, ground beef, chicken breast, cooked chicken, salmon, cheese, cantaloupe, and romaine lettuce. The food products were challenged with E. coli O157:H7 at ca. 3.0 log CFU/g and treated with the bacteriophage preparation at ca. 1 × 106, 5 × 106, or 1 × 107 PFU/g. Application of 5 × 106 and 1 × 107 PFU/g resulted in significant reductions (P < 0.05) in E. coli O157:H7 levels of up to 97% in all foods. When bacteriophages (ca. 1 × 106 PFU/g) were used to treat lower levels of E. coli O157:H7 (ca. 1 to 10 CFU/10 g) on beef chuck roast samples, mimicking the levels of STEC found under real-life conditions in food processing plants, the prevalence of STEC in the samples was significantly reduced (P < 0.05) by ≥80%. Our results suggest that this STEC-targeting bacteriophage preparation can result in significant reduction of both the levels and prevalence of STEC in various foods and, therefore, may help improve the safety and reduce the risk of recalls of foods at high risk for STEC contamination.

Bacteriophages Reduce Pathogenic Escherichia coli Counts in Mice Without Distorting Gut Microbiota.

We performed a study to (i) investigate efficacy of an Escherichia coli/Salmonella spp./Listeria monocytogenes-targeting bacteriophage cocktail (tentatively named F.O.P.) to reduce a human pathogenic E. coli strain O157:H7 in experimentally infected mice, and (ii) determine how bacteriophages impact the normal gut microbiota when compared with antibiotic therapy. A total of 85 mice were inoculated with E. coli O157:H7 strain Ec231 [nalidixic acid resistant (NalAcR)] via oral gavage, and were randomized into six groups separated into three categories: 1st category received PBS or No phage/No PBS (control), 2nd category received either F.O.P., F.O.P. at 1:10 dilution, or only the E. coli phage component of F.O.P. (EcoShield PXTM), and 3rd category received the antibiotic ampicillin. All therapies were administered twice daily for four consecutive days including before and after bacterial challenge; except ampicillin which was administered only before and after bacterial challenge on day 0. Fecal samples were collected at Days 0, 1, 2, 3, 5, and 10. Samples were homogenized and plated on LB plates supplemented with NalAc to determine viable Ec231 counts. Body weights were measured at every fecal sample collection point. qPCR was performed using specific E. coli O157:H7 primers to quantify the number of E. coli O157:H7 genome copies. Microbiota community profiles were analyzed using Denature Gradient Gel Electrophoresis (DGGE) and 16S rRNA sequencing. F.O.P. significantly (P < 0.05) reduced E. coli O157:H7 pathogen counts by 54%. Ampicillin therapy significantly (P < 0.05) reduced E. coli O157:H7 pathogen counts by 79%. Greater initial weight-loss occurred in mice treated with ampicillin (-5.44%) compared to other treatment groups. No notable changes in the gut microbiota profiles were observed for control and F.O.P. groups. In contrast, the antibiotic group displayed noticeable distortion of the gut microbiota composition, only partially returning to normal by Day 10. In conclusion, we found that F.O.P. administration was effective in reducing viable E. coli O157:H7 in infected mice with a similar efficacy to ampicillin therapy. However, the F.O.P. bacteriophage preparation had less impact on the gut microbiota compared to ampicillin.

A Bacteriophage Cocktail Eliminates Salmonella Typhimurium from the Human Colonic Microbiome while Preserving Cytokine Signaling and Preventing Attachment to and Invasion of Human Cells by Salmonella In Vitro.

Nontyphoidal Salmonella strains continue to be a major cause of foodborne illness globally. One intriguing approach to reducing the risk of salmonellosis is the direct ingestion of phages targeting Salmonella to enhance natural gut resilience and provide protection during foodborne disease outbreaks. We evaluated the ability of a prophylactically administered bacteriophage cocktail, the foodborne outbreak pill (FOP) targeting Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella, to resolve a Salmonella infection in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), a simulated gut platform populated by the human intestinal microbiome of healthy donors. The FOP preparation eliminated Salmonella enterica serovar Typhimurium from the colon compartment of the SHIME platform but health-associated metabolites, such as short-chain fatty acids and lactate, remained stable or increased in a donor-dependent manner. In studies of human intestinal cells, pretreatment of Salmonella Typhimurium with the FOP cocktail preserved lipopolysaccharide-stimulated signaling in a Caco-2-THP-1 Transwell system and prevented destruction of the Caco-2 monolayer by Salmonella. Adhesion and invasion of intestinal epithelial cells by Salmonella-a critical factor in Salmonella pathogenesis-was blunted when the bacteria were incubated with the FOP preparation before addition to the monolayer. The FOP phage cocktail was effective for (i) eliminating Salmonella from a simulated human gut without disturbing the indigenous microbiota and (ii) reducing the risk of invasion by Salmonella into the intestinal epithelia. These results suggest that the FOP preparation may be of value for reducing the risk of salmonellosis in humans, e.g., during foodborne disease outbreaks.

Bacteriophage Applications for Food Production and Processing.

Foodborne illnesses remain a major cause of hospitalization and death worldwide despite many advances in food sanitation techniques and pathogen surveillance. Traditional antimicrobial methods, such as pasteurization, high pressure processing, irradiation, and chemical disinfectants are capable of reducing microbial populations in foods to varying degrees, but they also have considerable drawbacks, such as a large initial investment, potential damage to processing equipment due to their corrosive nature, and a deleterious impact on organoleptic qualities (and possibly the nutritional value) of foods. Perhaps most importantly, these decontamination strategies kill indiscriminately, including many—often beneficial—bacteria that are naturally present in foods. One promising technique that addresses several of these shortcomings is bacteriophage biocontrol, a green and natural method that uses lytic bacteriophages isolated from the environment to specifically target pathogenic bacteria and eliminate them from (or significantly reduce their levels in) foods. Since the initial conception of using bacteriophages on foods, a substantial number of research reports have described the use of bacteriophage biocontrol to target a variety of bacterial pathogens in various foods, ranging from ready-to-eat deli meats to fresh fruits and vegetables, and the number of commercially available products containing bacteriophages approved for use in food safety applications has also been steadily increasing. Though some challenges remain, bacteriophage biocontrol is increasingly recognized as an attractive modality in our arsenal of tools for safely and naturally eliminating pathogenic bacteria from foods.

A bacteriophage cocktail targeting Escherichia coli reduces E. coli in simulated gut conditions, while preserving a non-targeted representative commensal normal microbiota.

Antibiotics offer an efficient means for managing diseases caused by bacterial pathogens. However, antibiotics are typically broad spectrum and they can indiscriminately kill beneficial microbes in body habitats such as the gut, deleteriously affecting the commensal gut microbiota. In addition, many bacteria have developed or are developing resistance to antibiotics, which complicates treatment and creates significant challenges in clinical medicine. Therefore, there is a real and urgent medical need to develop alternative antimicrobial approaches that will kill specific problem-causing bacteria without disturbing a normal, and often beneficial, gut microbiota. One such potential alternative approach is the use of lytic bacteriophages for managing bacterial infections, including those caused by multidrug-resistant pathogens. In the present study, we comparatively analysed the efficacy of a bacteriophage cocktail targeting Escherichia coli with that of a broad-spectrum antibiotic (ciprofloxacin) using an in vitro model of the small intestine. The parameters examined included (i) the impact on a specific, pre-chosen targeted E. coli strain, and (ii) the impact on a selected non-targeted bacterial population, which was chosen to represent a defined microbial consortium typical of a healthy small intestine. During these studies, we also examined stability of bacteriophages against various pH and bile concentrations commonly found in the intestinal tract of humans. The bacteriophage cocktail was slightly more stable in the simulated duodenum conditions compared to the simulated ileum (0.12 vs. 0.58 log decrease in phage titers, respectively). It was equally effective as ciprofloxacin in reducing E. coli in the simulated gut conditions (2-3 log reduction), but had much milder (none) impact on the commensal, non-targeted bacteria compared to the antibiotic.

Bacteriophage preparation lytic for Shigella significantly reduces Shigella sonnei contamination in various foods.

ShigaShield™ is a phage preparation composed of five lytic bacteriophages that specifically target pathogenic Shigella species found in contaminated waters and foods. In this study, we examined the efficacy of various doses (9x105-9x107 PFU/g) of ShigaShield™ in removing experimentally added Shigella on deli meat, smoked salmon, pre-cooked chicken, lettuce, melon and yogurt. The highest dose (2x107 or 9x107 PFU/g) of ShigaShield™ applied to each food type resulted in at least 1 log (90%) reduction of Shigella in all the food types. There was significant (P<0.01) reduction in the Shigella levels in all phage treated foods compared to controls, except for the lowest phage dose (9x105 PFU/g) on melon where reduction was only ca. 45% (0.25 log). The genomes of each component phage in the cocktail were fully sequenced and analyzed, and they were found not to contain any "undesirable genes" including those listed in the US Code for Federal Regulations (40 CFR Ch1). Our data suggest that ShigaShield™ (and similar phage preparations with potent lytic activity against Shigella spp.) may offer a safe and effective approach for reducing the levels of Shigella in various foods that may be contaminated with the bacterium.

Bacteriophages safely reduce Salmonella contamination in pet food and raw pet food ingredients.

Contamination of pet food with Salmonella is a serious public health concern, and several disease outbreaks have recently occurred due to human exposure to Salmonella tainted pet food. The problem is especially challenging for raw pet foods (which include raw meats, seafood, fruits, and vegetables). These foods are becoming increasingly popular because of their nutritional qualities, but they are also more difficult to maintain Salmonella-free because they lack heat-treatment. Among various methods examined to improve the safety of pet foods (including raw pet food), one intriguing approach is to use bacteriophages to specifically kill Salmonella serotypes. At least 2 phage preparations (SalmoFresh® and Salmonelex™) targeting Salmonella are already FDA cleared for commercial applications to improve the safety of human foods. However, similar preparations are not yet available for pet food applications. Here, we report the results of evaluating one such preparation (SalmoLyse®) in reducing Salmonella levels in various raw pet food ingredients (chicken, tuna, turkey, cantaloupe, and lettuce). Application of SalmoLyse® in low (ca. 2-4×106 PFU/g) and standard (ca. 9×106 PFU/g) concentrations significantly (P < 0.01) reduced (by 60-92%) Salmonella contamination in all raw foods examined compared to control treatments. When SalmoLyse®-treated (ca. 2×107 PFU/g) dry pet food was fed to cats and dogs, it did not trigger any deleterious side effects in the pets. Our data suggest that the bacteriophage cocktail lytic for Salmonella can significantly and safely reduce Salmonella contamination in various raw pet food ingredients.

Bacteriophage cocktail significantly reduces or eliminates Listeria monocytogenes contamination on lettuce, apples, cheese, smoked salmon and frozen foods.

ListShield™, a commercially available bacteriophage cocktail that specifically targets Listeria monocytogenes, was evaluated as a bio-control agent for L. monocytogenes in various Ready-To-Eat foods. ListShield™ treatment of experimentally contaminated lettuce, cheese, smoked salmon, and frozen entrèes significantly reduced (p < 0.05) L. monocytogenes contamination by 91% (1.1 log), 82% (0.7 log), 90% (1.0 log), and 99% (2.2 log), respectively. ListShield™ application, alone or combined with an antioxidant/anti-browning solution, resulted in a statistically significant (p < 0.001) 93% (1.1 log) reduction of L. monocytogenes contamination on apple slices after 24 h at 4 °C. Treatment of smoked salmon from a commercial processing facility with ListShield™ eliminated L. monocytogenes (no detectable L. monocytogenes) in both the naturally contaminated and experimentally contaminated salmon fillets. The organoleptic quality of foods was not affected by application of ListShield™, as no differences in the color, taste, or appearance were detectable. Bio-control of L. monocytogenes with lytic bacteriophage preparations such as ListShield™ can offer an environmentally-friendly, green approach for reducing the risk of listeriosis associated with the consumption of various foods that may be contaminated with L. monocytogenes.

Bacteriophage cocktail for biocontrol of Salmonella in dried pet food.

Human salmonellosis has been associated with contaminated pet foods and treats. Therefore, there is interest in identifying novel approaches for reducing the risk of Salmonella contamination within pet food manufacturing environments. The use of lytic bacteriophages shows promise as a safe and effective way to mitigate Salmonella contamination in various food products. Bacteriophages are safe, natural, highly targeted antibacterial agents that specifically kill bacteria and can be targeted to kill food pathogens without affecting other microbiota. In this study, we show that a cocktail containing six bacteriophages had a broadspectrum activity in vitro against a library of 930 Salmonella enterica strains representing 44 known serovars. The cocktail was effective against 95% of the strains in this tested library. In liquid culture dose-ranging experiments, bacteriophage cocktail concentrations of ≥10(8) PFU/ml inactivated more than 90% of the Salmonella population (10(1) to 10(3) CFU/ml). Dried pet food inoculated with a mixture containing equal proportions of Salmonella serovars Enteritidis (ATCC 4931), Montevideo (ATCC 8387), Senftenberg (ATCC 8400), and Typhimurium (ATCC 13311) and then surface treated with the six-bacteriophage cocktail (≥2.5 ± 1.5 × 10(6) PFU/g) achieved a greater than 1-log (P < 0.001) reduction compared with the phosphate-buffered saline-treated control in measured viable Salmonella within 60 min. Moreover, this bacteriophage cocktail reduced natural contamination in samples taken from an undistributed lot of commercial dried dog food that tested positive for Salmonella. Our results indicate that bacteriophage biocontrol of S. enterica in dried pet food is technically feasible.

Salmonella phages and prophages: genomics, taxonomy, and applied aspects.

Since this book was originally published in 2007 there has been a significant increase in the number of Salmonella bacteriophages, particularly lytic virus, and Salmonella strains which have been fully sequenced. In addition, new insights into phage taxonomy have resulted in new phage genera, some of which have been recognized by the International Committee of Taxonomy of Viruses (ICTV). The properties of each of these genera are discussed, along with the role of phage as agents of genetic exchange, as therapeutic agents, and their involvement in phage typing.

Bacteriophage administration significantly reduces Shigella colonization and shedding by Shigella-challenged mice without deleterious side effects and distortions in the gut microbiota.

We used a mouse model to establish safety and efficacy of a bacteriophage cocktail, ShigActive™, in reducing fecal Shigella counts after oral challenge with a susceptible strain. Groups of inbred C57BL/6J mice challenged with Shigella sonnei strain S43-NalAcR were treated with a phage cocktail (ShigActive™) composed of 5 lytic Shigella bacteriophages and ampicillin. The treatments were administered (i) 1 h after, (ii) 3 h after, (iii) 1 h before and after, and (iv) 1 h before bacterial challenge. The treatment regimens elicited a 10- to 100-fold reduction in the CFU's of the challenge strain in fecal and cecum specimens compared to untreated control mice, (P < 0.05). ShigActive TM treatment was at least as effective as treatment with ampicillin but had a significantly less impact on the gut microbiota. Long-term safety studies did not identify any side effects or distortions in overall gut microbiota associated with bacteriophage administration. Shigella phages may be therapeutically effective in a "classical phage therapy" approach, at least during the early stages after Shigella ingestion. Oral prophylactic "phagebiotic" administration of lytic bacteriophages may help to maintain a healthy gut microbiota by killing specifically targeted bacterial pathogens in the GI tract, without deleterious side effects and without altering the normal gut microbiota.

Evaluation of consumers' perception and willingness to pay for bacteriophage treated fresh produce.

Food-borne illnesses caused by bacteria such as enterohemorrhagic E. coli and Salmonella spp. take a significant toll on American consumers' health; they also cost the United States an estimated $77.7 billion annually in health care and other losses.1 One novel modality for improving the safety of foods is application of lytic bacteriophages directly onto foods, in order to reduce or eliminate their contamination with specific foodborne bacterial pathogens. The main objective of this study was to assess consumers' perception about foods treated with bacteriophages and examine their willingness to pay (WTP) an additional amount (10-30 cents/lb) for bacteriophage-treated fresh produce. The study utilized a survey questionnaire administered by telephone to consumers in 4 different states: Alabama, Georgia, North Carolina, and South Carolina. The results show that consumers are in general willing to pay extra for bacteriophage-treated fresh produce if it improves their food safety. However, income, race, and the state where a consumer lives are significant determinants in their WTP.