Introduction fuelled multiple biotechnological applications in very diverse

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Last updated: April 26, 2019

IntroductionEverydaypeople worldwide buy and consume a diversity of products of animal and plantorigin expecting these products to be safe. However, the current technologiesemployed to inactivate bacterial pathogens in foods are not infallible.Annually, millions of people become ill, are hospitalized, and die due to avariety of food borne pathogens, such as Salmonella, Staphylococcus aureus, Campylobacter, Escherichia coli, Listeria, transmitted throughfoods. The most e?cient means for limiting the growth of microbes are goodproduction hygiene, a rational running of the process line, and a well-designeduse of biocides and disinfectants.

Regardless of modern technologies, goodmanufacturing practices, quality control and hygiene contaminating bacteriastill can get access to food during slaughtering, milking, fermentation,processing, storage or packaging. Moreover, the extensive use of sanitizers andantibiotics has led to the development of resistant bacteria rendering theseprocedures less effective. Thus, to meet the primary goal of any food safetyprogram, the consumer protection, new food preservation techniques have to becontinually developed to meet current demands, in order to control the emergingpathogens and their impact at global scale (1).Researchon phage molecular biology has fuelled multiple biotechnological applications in very diversefields including bacterial detection systems, novel antimicrobials againstantibiotic-resistant bacteria, etc. Another promising field of application isthe use of phages as natural antimicrobials in food to inhibit undesirablebacteria (2). Bacteriophage descriptionA bacteriophage is a virus that infects and replicates within a bacterium.

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Bacteriophages arecomposed of proteins that encapsulate a DNA or RNA genome. Phages replicate within the bacterium following the injectionof their genome into its cytoplasm. To enter a hostcell, bacteriophages attach to specific receptors on the surface of bacteria.This specificity means a bacteriophage can infect only certain bacteria bearingreceptors to which they can bind, which in turn determines the phage’s hostrange. After making contact with the appropriate receptor, phage injectsgenetic material through the bacterial membrane. The host’s normal synthesis ofproteins and nucleic acids is disrupted, and it is forced to manufacture viralproducts instead. These products go on to become part of new virions within thecell, helper proteins that help assemble the new virions, or proteins involvedin cell lysis.

Phages may bereleased via cell lysis achieved by an enzyme called endolysin, which attacks andbreaks down the cell wall peptidoglycan (3).Bacteriophages are ubiquitous viruses, found whereverbacteria exist (soil, drinkable or sea water, even the intestines of animals). Themost outstanding advantage of phages is their harmless interaction toeukaryotic cells (human, animal, plants).  Bacteriophage applicationPhageswere discovered by Ernest Hankin (1896) and Frederick Twort (1915) whodescribed their antibacterial activity. However, Felix d’Herelle (1919) wasprobably the first scientist who used bacteriophages as a therapy to treat severedysentery. At that time, several companies then actively started up thecommercial production of phages against various bacterial pathogens for humanuse. However, phage production was quickly displaced by the discovery ofantibiotics.

Nowadays, the current threat of antibiotic-resistant bacteria hasrenewed the interest in exploring bacteriophages as biocontrol agents (1) (2).Phageshave a wide range of application: phage therapy, tools for detecting pathogens,water and food safety, agriculture and animal health. Bacteriophage-basedbiocontrol measurements have a great potential to enhance microbiologicalsafety based on relatively easy handling and their high and specificantimicrobial activity. The concept of combating pathogens in food can beaddressed at all stages of production in entire food chain. Bacteriophages aresuitable: (i) to prevent orreduce colonization and diseases in livestock (phage therapy). Phages have highspecificity to target specific bacteria determined by the cell wall receptorsand leave remaining microbiota untouched. (ii)to extend the shelf life of perishable manufactured foods as naturalpreservatives.

Low phages inherent toxicity is an advantage compered toantibiotics. (iii)to decontaminate carcasses and other raw products, such as fresh fruit andvegetables, and to disinfect equipment and contact surfaces (phagebiosanitation and biocontrol). Phages can generally withstand food processingenvironmental stresses (including food physiochemical conditions) (1) (2). Biocontrol of Escherichia coli usingbacteriophagesEscherichia coliis a gram-negative bacterium. SerotypeO157:H7 in particular, classi?ed as Shigatoxin-producing E. coli, is a well-known food poisoning pathogen. Thismicroorganism is highly virulent and a public health threat because ingestionof a concentration as low as 10-100 cells is able to cause infection (4) {2>15} Ruminants comprisethe principal reservoir for this strain and contamination of animal productsoccurs during milking or slaughtering (1). The most common route of E.

coli transmission to humans isvia undercooked contaminated food. To avoid food safety problemsbacteriophages therapy could be applied during preharvest or postharvestperiod.Postharvestinterventions have been e?ective in reducing E.

coli levels from fresh produce.Efficacy of bacteriophages against E.coli O157:H7 strain was tested on fresh cut lettuce surface by spay andimmerse application methods. Both methods provided a degree of protection fromintroduction of E. coli O157:H7 to fresh cut lettuce.

However,spray application of lytic bacteriophages was reported to be more effective inimmediately reducing E. coli O157:H7 populations on lettucesurfaces compared with immersion of lettuce in phage solutions (EcoShield™) (5). {9}Acocktail of three lytic phages specific for E. coli O157:H7(EcoShield™) or a control (phosphate buffered saline, PBS) was applied tolettuce in different ways.  (i)immersion of lettuce in 500 ml of EcoShield™ 8.

3 log PFU/ml or 9.8 log PFU/mlfor up to 2 min before inoculation with E. coli O157:H7.Phage-treated, inoculated lettuce pieces were stored at 4°C for and analyzedfor E. coli O157:H7 populations for up to 7 d. Immersion oflettuce in 9.8 log PFU/ml EcoShield™ for 2 min significantly (p < 0.

05)reduced E. coli O157:H7 populations after 24 h when stored at4°C compared with controls. (ii)spray-application of EcoShield™ (9.3 log PFU/ml) to lettuce after inoculationwith E. coli O157:H7 (4.10 CFU/cm2) followingexposure to 50 ?g/ml chlorine for 30 sec. Spraying technique was significantlymore effective in reducing E.

coli O157:H7 populations (2.22log CFU/cm2) on day 0 compared with control treatments (4.10 logCFU/cm2) (5).  {9}Preharvestapplication of phages to poultry by aerosol spraying and intramuscular injectionmethods has been successful to prevent fatal respiratory infections in broiler chickens (6). {2>16} However, phage administration via addition to bird drinking water proved to be ine?cient in protecting the birds from fatal E.

coli respiratory infections (7).{2>17}The main speculated causes for the failure of oraltreatments have been reported to be nonspeci?c binding of phages to food particles and other debris in the rumen and gastrointestinal tract (8). {2>18} Biocontrol of Staphylococcus aureus using bacteriophages Staphylococcus aureusis a gram-positive bacterium, is considered to be a major threat to foodsafety.  S. aureus can causetoxin-mediated diseases within 1 to 6 h after consumption of contaminatedfoods.

Staphylococcus aureus also isthe most common agent of mastitis in dairy cows (1). {2} Mastitis caused by Staphylococcus aureus isa major concern to the dairy industry due to its resistance to antibiotictreatment and its propensity to recur chronically (9){10}. The ability of lytic S. aureus bacteriophageK to eliminate bovine S. aureus intramammaryinfection during lactation was evaluated in a placebo-controlled, multisitetrial. Results revealed that phage treatment was not effective againstpreexisting subclinical S.

aureus mastitis.The efficacy of bacteriophage in the treatment appeared to be limited under thetreatment conditions studied. Natural inhibitory effect of raw milk causedphage inhibition and treatment failure. Moreover, in healthy lactating cowsphage therapy even elicited a large increase in the milk somatic cell count (9).

{10}PhageK inactivation was also reported in raw milk. The interaction of bacteriophage K and S. aureus strain Newbould 305 was studied in rawbovine whey and serum. Incubation of S. aureus withphage in whey showed that the bacteria is more resistant to phage lysis whengrown in whey or bovine serum. The adsorption of whey proteins to the S. aureus cell surface appeared to inhibit phageattachment and thereby hindered lysis (10).

{11}However,a cocktail of two lytic phages of dairy origin produced a complete elimination of the pathogen inultra-high-temperature (UHT) whole milk at 37 °C.This indicates that lytic bacteriophages could beused as biopreservatives when natural inhibitory milk propertiesare removed (11).{12} Biocontrol of Salmonella using bacteriophages Salmonella,is a genus of gram-negative facultative intra-cellular species, is consideredto be one of the principal causes of zoonotic diseases reported worldwide. Salmonella serovars can colonize andpersist within the gastrointestinal tract, and so human salmonellosis iscommonly associated with consumption of contaminated foods of animal origin ortransmitted via contaminated water. Salmonellais also a known spoilage bacterium in processed foods.

Once ingested, thismicroorganism can cause fever, diarrhea, abdominal cramps, and evenlife-threatening infections (1){2}.Salmonella biocontrolefficacy of bacteriophage FO1-E2 was evaluated. Collected data showed that virulentphages such as FO1-E2 offer an effective biocontrol measure for Salmonella infoods. Experiment conducted at 8 °C storage temperature reported no viablebacteria detection after treatment. However, at 15 °C storagetemperature, Salmonella growth was detected but suppressedby at least 2 and up to 5 log units on different foods. Results prove thatbacteriophage FO1-E2 is an effective biocontrol agent for Salmonella (12). {3} Materialsand methods:Hotdogs, cooked and sliced turkey breast, mixed seafood, chocolate milk, and eggyolk (pasteurized) were used for experiment.

After screening for contaminationwith Salmonella, foods were stored frozen at – 80 °C until use(except egg yolk, which was used fresh).Threefood samples were required for each experiment: (i) negative control containingno bacteria, (ii) positive control containing Salmonella Typhimurium only, and (iii) samplecontaining Salmonella Typhimuriumand bacteriophage FO1-E2. The concentration of Salmonella in the food sample were based on inoculum correspondingto approximately 1% of the total sample size. Before addition of phage, foodsamples were pre-incubated at 8 °C or 15 °C for 1–2 h to allowthe bacteria to adapt to the conditions.

Phage FO1-E2 was then added at aconcentration of 3 × 108 pfu/g or ml. Samples werefurther incubated at 8 °C or 15 °C, for up to 6 days. Bacterialand phage counts were determined by duplicate plating, and all food experimentswere independently performed at least twice. As a result, determination ofviable counts (cfu) from the food samples was straightforward (12). {3}Food treated with phageand incubated at 8 °C showed no viable cells could be recovered by directplating after 1 day of incubation, corresponding to a reduction ofapproximately 3 logs (12).{3}During incubation at 15 °Capplication of FO1-E2 resulted in a Salmonella viable countdecrease of at least 2 logs in the first two days, which was followed bysome regrowth during the remaining incubation period. After six days, viablecounts were more than five orders of magnitude lower in chocolate milk and onsliced cooked turkey breast (p < 0.05).

On hot dogs, Salmonella numberswere reduced by 3.0 logs (p < 0.05), and on mixed seafood by1.9 logs (p < 0.05). In egg yolk, a significant difference tothe control samples was observed after two days (2.6 log units,p < 0.05), but not after five or six days of incubation(p > 0.

05) (12).{3}Moreexamples of successful phage application against Salmonella were reported. The activityof the Salmonella phage SJ2 was tested in cheddar cheesemanufacturing (13).  {13} In the presence of phages (MOI 104), Salmonella didnot survive in the pasteurized cheeses after 89 days.

In another study,Salmonella phage cocktails have been assayed on fruits. Phage numbers remainedrelatively stable on melon and gave a significant reduction of target bacteria.On the contrary, a quick decline of infective phage particles was observed onapples due to the lower pH of this fruit (14).{14} ConclusionPhagescould be successfully applicated in food industry to solve major food safetyproblems.

Phages are harmless to human cells and do not secrete toxiccompounds, expose only targeted bacteria, thus, natural microbiota is notexterminated. Phage therapy could be used to both preharvest and postharvestapplication. Controlof E.

coli, Salmonella and S. aureus was explored in this review. E. coli control by bacteriophages onfresh cut lettuce surface and respiratory infections in broiler chickensproved to be advantageous. However, phages treatment has not succeeded to healmastitis caused by S.

aureus.Interaction with other molecules could reduce phage performance. On the otherhand, Salmonella growth rate wassignificantly reduced in most of tested ready to eat (RTE) foods both at 8 °Cand 15 °C incubation temperatures. Cheddar cheesemanufacturing control was successful using phages and Salmonella was not detected.

In conclusion, phage therapyhas a great potential to solve food safety problems caused by microbiologicalcontamination.  

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