CC BY
0Hindawi Publishing Corporation International Journal of Microbiology Volume 2012, Article ID 863945, 13 pages doi:10.1155/2012/863945
Review Article
Bacteriophages and Their Role in Food Safety
Sanna M. Sillankorva, Hugo Oliveira, and Joana Azeredo
Institute for Biotechnology and Bioengineering (IBB), Centre for Biological Engineering, University ofMinho, Campus de Gualtar, 4710-057 Braga, Portugal
Correspondence should be addressed to Joana Azeredo, jazeredo@deb.uminho.pt Received 23 August 2012; Accepted 31 October 2012 Academic Editor: Beatriz Martinez
Copyright © 2012 Sanna M. Sillankorva et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The interest for natural antimicrobial compounds has increased due to alterations in consumer positions towards the use of chemical preservatives in foodstuff and food processing surfaces. Bacteriophages fit in the class of natural antimicrobial and their effectiveness in controlling bacterial pathogens in agro-food industry has led to the development of different phage products already approved by USFDA and USDA. The majority of these products are to be used in farm animals or animal products such as carcasses, meats and also in agricultural and horticultural products. Treatment with specific phages in the food industry can prevent the decay of products and the spread of bacterial diseases and ultimately promote safe environments in animal and plant food production, processing, and handling. This is an overview of recent work carried out with phages as tools to promote food safety, starting with a general introduction describing the prevalence of foodborne pathogens and bacteriophages and a more detailed discussion on the use of phage therapy to prevent and treat experimentally induced infections of animals against the most common foodborne pathogens, the use of phages as biocontrol agents in foods, and also their use as biosanitizers of food contact surfaces.
1. Introduction
Everyday people worldwide buy and consume a diversity of products of animal and plant origin expecting these products to be safe. However, annually, millions of people become ill, are hospitalized, and die due to a variety of foodborne pathogens transmitted through foods. For instance, the World Health Organization estimated in their fact sheet of March 2007 (no. 237) that in 2005, over 1.8 million people died due to diarrhoeal diseases. Furthermore, annually in the USA alone, there are roughly 48 million illnesses, 128,000 hospitalizations, and even 3,000 deaths caused by foodborne pathogens [1].
Regardless of modern technologies, good manufacturing practices, quality control and hygiene, changes in animal husbandry, agronomic process, and in food or agricultural technology, food safety is continuously challenged by changes in lifestyle and consumer demands (e.g., ready-to-eat products) and also by the increase of international trade [2]. The most efficient means for limiting the growth of microbes are good production hygiene, a rational running
of the process line, and a well-designed use of biocides and disinfectants [3]. However, even when acceptable cleaning procedures are applied, bacteria are found in foods and food contact surfaces [4]. Food products may become contaminated at different stages along the food chain, from growth or production until the final consumption. Furthermore, the inherent ability of pathogens to attach to living and inert surfaces, where they start living in microbial communities known as biofilms, and become highly tolerant to varied antimicrobial agents [5] also contributes to the pathogen prevalence in foods and food contact surfaces. So, to meet the primary goal of any food safety program, the consumer protection, new food preservation techniques have to be continually developed to meet current demands, in order to control the emerging pathogens and their impact at global scale.
2. Bacteriophage: Charting the Path to Food Safety
(Bacterio)phage, viruses specifically infecting bacteria, are harmless to humans, animals, and plants. Since the discovery
of phages in 1915, they have been extensively used not only in human and veterinary medicine but also in various agricultural settings. Being obligatory parasites, upon multiplication by taking over host protein machinery, phages can either cause cell lysis to release the newly formed virus particles (lytic pathway) or lead to integration of the genetic information into the bacterial chromosome without cell death (lysogenic pathway).
Towards a food safety perspective, strictly lytic phages are possibly one of the most harmless antibacterial approaches available.
Phages offer advantages as biocontrol agents for several reasons: (i) high specificity to target their host determined by bacterial cell wall receptors, leaving untouched the remaining microbiota, a property that favors phages over other antimicrobials that can cause microbiota collateral damage; (ii) self-replication and self-limiting, meaning that low or single dosages will multiply as long as there is still a host threshold present, multiplying their overall antimicrobial impact; (iii) as bacteria develop phage defense mechanisms for their survival, phages continuously adapt to these altered host systems; (iv) low inherent toxicity, since they consist mostly of nucleic acids and proteins; (v) phages are relatively cheap and easy to isolate and propagate but maybecome time consuming when considering the development of a highly virulent, broad-spectrum, and nontransducing phage; (vi) they can generally withstand food processing environmental stresses (including food physiochemical conditions); (vii) they have proved to have prolonged shelf life. Phages are readily abundant in foods and have been isolated from a wide variety of raw products (e.g., beef, chicken) [6, 7], processed food (e.g., pies, biscuit dough, and roast turkey) [8], fermented products (e.g., cheese, yoghurt) [9], and seafood (e.g., mussels and oysters) [8, 10]. This suggests that phages can be found in the same environments where their bacterial host(s) inhabit, or once were present and that phages are daily consumed by humans. Furthermore, the use of antibiotics prophylactically and therapeutically in farm animals has become a major concern due to their possibility of contributing to the declining efficacy of the antibiotics used to treat bacterial infections in humans and leading to the alarming emergence of superbugs like Salmonella DT104 and the methicillin-resistant and multidrug-resistant Staphylococcus aureus. The use of phages to promote food safety can be basically done at four different stages along the food chain (Figure 1).
Reduction of pathogens colonization in animals during primary production (phage therapy) is a strategy followed in primary production just before slaughter or during animal growth to reduce the probability of cross-contamination with the animal feces during food processing. For example, it is estimated that a reduction of 2 log on the Campylobacter loads in poultry intestines is sufficient to diminish 30fold the incidence of campylobacteriosis associated with consumption of chicken meals [11]. The proof of principle of phage therapy in animals was already established for several pathogens (detailed description below). Phages can be administered orally, incorporated in drinking water or food, to control Salmonella and Campylobacter in poultry,
or by spray to target avian pathogenic E. coli in poultry, and orally/rectally to control E. coli in ruminants.
Reduction of colonization on foods (biocontrol) during industrial food processing can be accomplished by applying phages directly on food surfaces, for example, in case of meats, fresh produce, and processed foods or even mixed onto raw milk. Experimental data reveals that phages are very effective against actively growing bacteria and lose effectiveness in nongrowing bacteria [12]. In these cases, effective control could be achieved, applying high titres of phages to control pathogens by "lysis from without" mechanisms [6, 13] or whenever phages start to replicate immediately after the food begins to warm (i.e., during preparation, handling, and/or consumption).
In food industry, biofilms are found on the surfaces of equipment used, for example, in food handling, storage, or processing, especially in sites that are not easy to clean or to sanitize. Some of the work using phages against in vitro biofilms formed by spoilage and pathogenic bacteria show that under ideal conditions significant viable cell reductions are achieved and thus, their use for biosanitation is promising although very challenging due to the diversity of bacteria found in different settings.
Phages are also excellent as food biopreservation agents since they are reported to lyse hosts at temperatures as low as 1°C [14, 15], limiting growth of pathogenic and spoilage bacteria on refrigerated foods (specially psychrotrophic bacteria); once the foods are taken to room temperature, phages can further control their proliferation [16].
3. Phages Targeting Different Food Pathogens
The global incidence of foodborne disease and costs associated are difficult to estimate, however cost at least $7 billion dollars each year in medical expenses and lost productivityin the United States according to the United States Department of Agriculture's Economic Research Service [17]. The actual figure is higher since this estimate reflects illnesses caused only by the major foodborne pathogens. The most frequently reported pathogens from animal origin, responsible for such impact, are separately discussed in this section, focusing on their contamination sources and their harms for animals and humans and furthermore summarizing the recent phage interventions and main outcomes. We also briefly address some of the common phytopathogens and discuss phage applications carried out in an attempt to decrease crop diseases and product loss.
4. Foodborne Pathogens from Animal Origin
The four main foodborne pathogens from animal origin are accounted to be E. coli, Campylobacter, Salmonella, and Listeria. These bacteria are all common contaminants of ruminants, poultry, and swine and are usually carried in their gastrointestinal tract asymptomatically. Research on the use of phages against foodborne pathogens from animal origin has mainly focused on the optimization of preharvest interventions where the phage administration routes and
• Reduction of colonization in living animals during primary production
• Disinfection of food contact surfaces and equipments
Therapy
Sanitation
Phage applications
Control
Preservation
• Reduction of colonization of foods at industry food processing
• Prevention of contamination and pathogen proliferation on foods during storage and marketing of the final products
Figure 1: Feasible applications of phages along the food chain towards an increased food safety (adapted from Greer [18]).
delivery processes have received most attention and also on the optimization of postharvest strategies. The usage of phages as a preharvest strategy is made directly by administering phages to livestock to prevent animal illness and/or also to minimize the pathogen carriage in the gastrointestinal tract, thereby preventing pathogen entry to the food supply. Postharvest strategies are based on the use of phages directly on animal carcasses in an attempt to sanitize the products.
Escherichia coli is a gram-negative bacterium. Serotype O157:H7 in particular, classified as Shiga toxin-producing E. coli, is a well-known food poisoning pathogen. Its major reservoir comprises ruminants and, as it can survive well under intestinal conditions, if proper care is not taken during slaughter, the contents of the intestines, fecal material, or dust on the hide may contaminate meats [19]. The most common route of E. coli transmission to humans is via undercooked contaminated food, while water and raw milk are assumed to be related to cross-contamination events, by direct or indirect contact with feces. This microorganism is highly virulent and a public health threat because ingestion of a concentration as low as 10 cells is able to cause infection [1,20,21].
Recent pre- and postharvest phage research targeting E. coli is listed in Table 1.
Recent phage therapy to decrease E. coli levels on farm animals has focused mainly on poultry and ruminants.
Application of phages to poultry has been successful to prevent fatal respiratory infections in broiler chickens [2224]. Several different approaches have been used; however, aerosol spraying and intramuscular (i.m.) injection have given the best results and reduced significantly the mortality
of broiler chicken. Despite these results, phage administration via addition to bird drinking water proved to be inefficient in protecting the birds from fatal E. coli respiratory infections.
Although some successful results of phage therapy in ruminants have been reported using oral delivery of phages via direct administration or addition to drinking water and/or feed, the majority of the recently published papers suggest that oral treatment is unsuccessful in reducing E. coli levels (see Table 1).
The main speculated causes for the failure of oral treatments have been reported to be (i) nonspecific binding of phages to food particles and other debris in the rumen and gastrointestinal tract [25]; (ii) phage inactivation upon contact with the acidic conditions of the abomasum [26]; (iii) causing an insufficient number of orally phages reaching the gastrointestinal tract [27]. An interesting approach to reduce coliphage inactivation has been described by Stanford and colleagues in 2010. These authors successfully encapsulated phages in polymeric matrices which resisted in vitro acidic conditions and furthermore, once delivered orally to steers caused reduction of E. coli levels [28].
Essentially, oral delivery of phages has reported to be successful either using a cocktail of phages or combined with rectal treatment. For instance, a combined oral/rectal treatment using phages KH1, SH1 was able to reduce E. coli levels compared to oral treatment alone or to individual phage administration, but still this combined oral/rectal treatment using a phage cocktail did not cause total eradication of the pathogen from cattle [32]. Also, a cocktail of phages CEV1 and CEV2 were reported to lead to more than 99.9% of reduction of E. coli in sheep guts [36]. However, although reductions of E. coli levels in different
Year Animal/product Phage(s) Strategy Main outcome Refs
Preharvest application
2002 Poultry (broiler chicken) 2002 Poultry (broiler chicken)
SPR02 and DAF6
2003 Poultry (broiler chicken) SPR02 and DAF6
2003 Ruminant (lamb) 2006 Poultry (broiler chicken) 2006 Ruminant (sheep)
2006 Ruminant (cattle)
2009 Ruminant (steer)
2010 Ruminant (steer)
2010 Poultry
2010 Ruminant (cattle)
2011 Ruminant (sheep)
SPR02 and DAF6 CEV1
Phage cocktail (KH1, SH1)
Phage cocktail
Phage cocktail (wV8, rV5, wV7, wV11)
Phage cocktail
Phage cocktail (e11/2, e4/1c)
Phage cocktail (CEV1, CEV2)
Air sac or drinking Air sac inoculation prevented mortality. Drinking water [24] offered no protection
Significant decrease of mortality but not complete protection
Aerosol spray effective only when applied immediately after bird challenge with E. coli. A single i.m. injection reduced mortality when applied immediately and 24 and 48 h after challenge No reduction of fecal shedding over 30 days
water Sprayed
Sprayed and i.m. injection
Oral delivery i.m. injection into the left thigh Oral delivery
Only high phage titers (108) reduced mortality
2 log CFU reduction within 2 days Oral/rectal delivery No reduction of CFU when applied orally. Combined (via drinking oral/rectal treatment reduced CFU but did not
water) eradicate it
_ ,, , , Small fecal shedding reduction of oral/rectal compared
Oral/rectal delivery
to the rectal treatment and control
Oral delivery (gelatin capsules and in feed) Oral delivery and spray
Oral delivery Oral delivery
[33] [28]
No reduction of fecal shedding of nalidixic acid-resistant E. coli O157: H7, but duration of shedding was reduced by 14 days Significant reduction of mortality in large scale animal experiments
Rapid CFU decrease within 24 to 48 h, but no decrease in fecal shedding levels Cocktail eradicated (>99.9%) the pathogen and is more [36] effective than CEV1 alone
Postharvest application
2004 Meat e11/2, e4/1c, pp01 Applied on top Eradication in seven of nine samples [37]
2008 Fresh produce (tomato, spinach) and meat Phage cocktail (ECP-100) Applied on top/sprayed 94% and 100% reductions in CFU after 120 h and 24 h in tomato and spinach; 95% reduction in ground meat after 24 hat 10°C [38]
2009 Fresh produce (lettuce, cantaloupe) Phage cocktail (ECP-100) Sprayed Added to foods Significant CFU reductions after 2 days at 4° C [39]
2011 Fresh produce (lettuce, spinach) Phage cocktail (BEC8) together with trans- cinnamaldehyde (TC) No survivors detectable after 10 min of phage combined with the TC treatment [40]
2011 Food surfaces (spinach blades) Phage cocktail Sprayed 4.5 log reduction CFU after 2 h of phage [41]
2011 Food surfaces (steel, ceramic chips) Phage cocktail (BEC8) Applied on top Eradication after 10 min at 37°C and after 1 h at 23°C [40]
organs have been described, phages still fail in reducing fecal shedding [29, 35], and only in one published work the authors managed to reduce fecal shedding duration by 14 days [28]. In 2008, Niu and colleagues investigated two different sampling techniques: fecal grab and rectoanal mucosal swab for surveillance of E. coli O157:H7 [42] and to study the role of phage as a mitigation strategy. Their study showed discrepancy between fecal and rectoanal mucosal swab sampling in 63 of the 213 positive samples from experimentally inoculated feedlot steer. This shows that sampling procedures can significantly influence the merit of
phage and that prudence must be taken to validate phage therapy for E. coli O157:H7 control [42].
All postharvest interventions reported since 2000 have been effective in reducing E. coli levels from fresh produce and meats. Also, phage application to E. coli contaminated food contact surfaces has led to significant reductions proving that phage can be used safely for equipment and food contact surface sanitation.
The successful use of coliphages has led to the development of a phage-product (EcoShield (Intralytix)) which received regulatory approval from the Food and Drug
Administration (FDA) in 2011. This product can be used on red meat parts and trims intended to be ground (Food Contact Notification no. 1018) and has proved to eliminate from 95 to 100% of E. coli O157:H7. Finalyse (Elanco Food Solutions) is another product on the market using naturally occurring phages specific for E. coli O157:H7. Finalyse is sprayed on cattle to reduce the load of E. coli, prior to its entering the beef packing facility (preharvest strategy).
Campylobacter is a genus of gram-negative, spiral, motile, and microaerophilic bacteria with an optimal growth temperature around 41 °C. C. jejuni and C. coli members are considered to be major aetiological agents of enteric diseases worldwide. Campylobacter is the most commonly reported zoonosis in Europe (EFSA 2011), and C. jejuni, in particular, is estimated to cause approximately 845,000 illnesses, 8,400 hospitalizations, and 76 deaths each year in the USA [1]. This widespread infection is explained because ingestion of low doses (400-500 cells) [43] can cause campylobacteriosis typically characterized by fever, bloody diarrhea, and acute abdominal pain [44]. Campylobacter is capable of colonizing the intestine of poultry and cattle, and thus infection is mostly acquired by fecal-oral contact, ingestion of contaminated foods (i.e., raw meat and milk contaminated through feces), and waterborne through contaminated drinking water [45-47]. The widespread disease and economic impact on agriculture and food industries has led to the development ofvarious approaches to contain this infection using bacteriophages (Table 2).
Phage preharvest interventions reported so far have been successful in reducing Campylobacter numbers in the cecal content and feces of experimentally infected broilers and have not caused any adverse health effects. However, some degree ofresistant phenotypes, recovered from phage-treated chickens, has been reported [48-50], of which some were found to display clear evidence of genomic rearrangements [51]. Scott and colleagues demonstrated that Campylobacter virulent phages have the potential to activate dormant prophages, leading to rapid pathogen evolution towards the development of phage-resistant phenotypes. These authors also showed that whilst pathogen evolution can be rapid, resistance to the therapeutic bacteriophage is associated with a decreased fitness to environment, specifically meaning that phage-resistant phenotypes exhibited a decreased ability to colonize the gastrointestinal tract [51]. These genomic instability findings suggest that C. jejuni adopt this strategy to temporarily survive local environmental pressures, including phage predation and competition for resources. The key elements for the success of phage therapy against Campylobacter in broiler chickens are a proper selection of phage, the dose of phage applied, and the time elapsed after administration [48].
As for postharvest strategies, two studies have been reported (see Table 2) in which phages reduced Campylobacter contamination following a "lysis from without" mechanism. This suggests that Campylobacter phages can be used as a tool for biocontrol purposes.
It is known that Campylobacter attaches and forms biofilms on surfaces as a measure to overcome environmental stresses, such as aerobic conditions, desiccation, heating,
disinfectants, and acidic conditions frequently encountered in food environments [52]; however, to date, we were only able to find one report evaluating the efficacy of Campylobacter phages of disrupting biofilm formed on glass. In this study, phages were able to reduce by 1 to 3 log the viable cell counts under microaerobic conditions; however, after treatment above 84% of the surviving bacteria were resistant to the two phages applied [53].
Salmonella, is a genus of gram-negative facultative intra-cellular species, is considered to be one of the principal causes of zoonotic diseases reported worldwide. Salmonella serovars can colonize and persist within the gastrointestinal tract, and so human salmonellosis is commonly associated with consumption of contaminated foods of animal origin. Salmonella infections cost nearly 3 million euros in EU per annum in health care systems [17, 54]. Salmonella enterica serovars, Enteritidis and Typhimurium, are responsible for the majority of Salmonella outbreaks, and most events are reported to be due to consumption of contaminated eggs and poultry, pig, and bovine meats, respectively [55]. Salmonella is also a known spoilage bacterium in processed foods. Once ingested, this microorganism can cause fever, diarrhea, abdominal cramps, and even life-threatening infections [56]. To prevent such infections, a number of studies on animal phage therapy have been reported where phages were used to prevent or reduce colonization and diseases in livestock. All recent studies are summarized in Table 3.
Phage therapy of experimentally Salmonella-infected poultry and swine animals has been successful and significantly decreased Salmonella in major tissues such as ileum and cecal tonsils. Although these results have been mostly obtained within contrived laboratory conditions, the success of postslaughter phage employment, to lower the risk of cross-contamination, will only be determined after extending these studies to poultry farms.
Apart from one report in 2001, where the multivalent Felix01 phage was used, all other in vivo experiments performed recently were carried out using cocktails of two to six phages. Furthermore, Ma and colleagues (2008) have encapsulated phage Felix01 in a chitosan-coated Ca-alginate spheres [57] and have found in in vitro studies that this technique preserves phage viability upon exposure to acidic conditions; however, to our knowledge, these encapsulated phages have not yet been tested in vivo. Valuable outputs have been made in the last couple of years in terms of using phages for Salmonella biocontrol. Today, two phage-products are available: (1) BacWash from OmniLytics Inc. which received, in 2007, USDA's Food Safety and Inspection Services approval to be commercialized and applied as a mist, spray, or wash on live animals prior to slaughter; (2) BIO-TECTOR S1 phage product from CheilJedang Corporation that is to be applied on animal feed to control Salmonella in poultry.
Unlike phage preharvest strategies on animals, several postharvest strategies have adopted the use of only one phage and not a cocktail. All Salmonella phages reported have been able to decrease the number of viable cells present on raw meats, processed and ready-to-eat foods, and fresh produce. Furthermore, the combined treatment of phage
Year Animal/product Phage(s) Strategy Main outcome Refs
Preharvest application
2005 Poultry (broiler chickens) $CP8, $CP34 Oral delivery in antacid suspension Decrease of CFU between 0.5 and 5 log CFU/g in the cecal content over a 5-day period posttreatment [48]
2005 Poultry (broiler chickens) 69, 71 Oral delivery Reduction of CFU by 1 log within 5-day period post-treatment. Phage preventive treatment caused a delay in a colonization [58]
Poultry (broiler chickens) Reduction of 2 log CFU per g in cecal content after 48 h
2009 CP220 Oral delivery inoculated C. jejuni and C. coli birds with a single dose (7 log PFU) of CP220 [49]
2010 Poultry (broiler chickens) Phage cocktail Oral delivery (oral gavage and in feed) Reduction levels of C. coli and C. jejuni in feces by 2 log CFU per g when administered by oral gavage and in feed [50]
Postharvest application
2003 Meat (chicken skin) $2 Applied on top 1 and 2 log CFU reduction at 4°C using 107 PFU per mL. 105 and 103 PFU per mL failed to decrease CFU [58]
2003 Meat (chicken skin) $29C Applied on top MOI 1 caused less than 1 log reduction in CFU; MOI 100-1,000 caused 2 log reductions in CFU [49]
2008 Meat (raw and cooked beef) Cj6 Applied on top The largest reductions were recorded at high host cell density on both raw and cooked beef over a period of 8 days incubation at 51°C [16]
and Enterobacter asburiae, a strain exhibiting antagonistic activity against Salmonella, to control this pathogen on tomatoes, mung bean sprouts, and alfalfa seeds, represents a highly promising, chemical-free approach. However, in some settings phages were found to be readily immobilized by the food matrix and, although retaining infectivity, they lost the ability to diffuse and infect target cells [59].
Listeria monocytogenes is a gram-positive, motile, and facultative intracellular bacterium, that can grow under several food matrices and storing conditions (e.g., high salt levels, low pH, lack of oxygen, and low temperatures) [60]. Invasive infection by L. monocytogenes causes listeriosis and is transmitted to humans with 103 CFU/mL levels. It is often associated with contaminated minimally processed food such as ready-to-eat (RTE) products, poultry, and dairy products or related to cross-contamination after the heat treatment process of foods stored at low temperatures [61, 62]. Despite its low incidence, estimated to be in order of 2 of 10 reported cases per million per year in Europe [63], its pathogenicity causes a high mortality rate of approximately 255 deaths each year in the USA alone according to the Centers for Disease Control and Prevention (CDC) [1]. As so, contingency measures have led to the establishment of a limit for common RTE foods of 100 CFU per gram in EU and a zero tolerance policy in the USA.
Considering the sources of Listeria outbreaks, phage research has focused on postharvest applications (Table 4).
The first two studies in Table 4 were carried out using a combination of phage and a nisin, a broad spectrum antibacteria peptide used during production to extend shelf life by suppressing gram-positive spoilage and pathogenic bacteria. While in ground beef the phage-nisin combination revealed to be ineffective, this strategy had a synergistic effect once added to melon and apple resulting in an improved reduction of Listeria compared to phage or nisin alone. The efficacy
of phage-nisin mixture was however significantly reduced in apples on the account of a decline of phage numbers possibly due to the low pH. Phage biocontrol should therefore be optimized separately for each food matrices under study. Four other studies have used phage P100, which was highly effective in inhibiting Listeria growth at storage temperatures for several days (see Table 4). Like in Campylobacter, only one article, by Soni and Nannapaneni (2010), on the efficacy of phages against biofilms was found for Listeria monocytogenes. These authors evaluated the ability of P100 against 21L. monocytogenes strains belonging to 13 serotypes and found that P100 reduced by 3.5 to 5.4 log/cm2 the viable cells present in stainless steel surfaces [64]. Nonetheless, studies carried out on a variety of experimentally contaminated meats, fresh produce, and processed food among others show that biocontrol is influenced by phage contact time and phage dose, regardless of higher or lower temperature. Despite this fact, the results conducted on RTE foods and meats using LMP-102 phage preparation (six-phage cocktail) allowed the commercialization of ListShield phage product from Intralytix. Also, LISTEX P100 phage-based-product (from EBI Food Safety) is being commercialized with a GRAS status (generally recognized as safe) to prevent Listeria contamination on food products and food processing facilities.
Staphylococcus aureus is a gram-positive bacterium, is considered to be a major threat to food safety [1, 90], and also is the most common agent of mastitis in dairy cows [91]. CDC estimates that staphylococcal food poisoning, which results from the consumption of foods containing sufficient amounts of one or more preformed enterotoxins [91, 92], is 242.148 cases annually in the USA [1]. The mechanisms described for the contamination of foods with S. aureus can be of animal or human origin, such as due to the infection and colonization of livestock or farm workers and even
Year Animal/product
Phage(s)
Strategy
Main outcome
Preharvest application
Poultry (chicken) Swine (pig) Poultry
(broiler chickens)
Poultry (chickens)
Poultry
(broiler chickens) Poultry
(broiler chickens)
Poultry (chickens)
Swine (pig) Swine
(weaned pigs)
Poultry
(chickens)
Phage cocktail
Felix01
CNPSA1, CNPSA3, CNPSA4
Phage cocktail (Sa2,S9, S11)
$151, $25, $10
Phage cocktail (CB40, WT450)
Phage cocktail
Phage cocktail Phage cocktail
Oral delivery (direct and via feed)
Oral delivery and i.m.
Oral delivery
Oral delivery phage/competitive exclusion Oral delivery (antacid suspension)
Oral delivery
Oral delivery (coarse
spray/drinking water)
Oral delivery
Oral delivery (via feed)
Reduction of CFU in cecal counts between 0.3 and 1.3 log compared to controls birds
Reduction of CFU in the tonsils and cecum
Reduction of CFU by 3.5 orders of magnitude after five days
Reduction of CFU in cecum and ileum after phage cocktail and/or competitive exclusion treatment
Reduction of 4.2 log and 2.19 log with phages $151 and $25 within 24 h compared with control Reduction of CFU in cecal tonsils after 24 h. No significant differences at 48 h compered to controls
Reduction of intestinal colonization often-day-old experimentally contaminated birds
Reduction of colonization by 99.0 to 99.9% in the tonsils, ileum, and cecum
Significant reduction of CFU in the rectum
Phage prevented horizontal transmission on six-week-old infected chickens
Postharvest application
2001 Processed food (ripened cheese) Fresh produce SJ2 Added to milk No survival during 89 days in pasteurized cheeses containing phages (MOI 104) [75]
2001 (fresh-cut melon and apple) Phage cocktail Added to foods Significant CFU reduction on melon but not on apple [76]
Meat (chicken skin) Meat MOI 1 caused less than 1 log reduction in CFU; MOI
2003 P22, 29C Applied on top 100-1,000 caused 2 log reductions in CFU and eradicated resistant strains [13]
2003 (chicken frankfurters) Felix O1 Approx. 2 log reduction with a MOI of 1.9 X 104 [21]
2004 Fresh produce (sprouting seeds) A, B Applied by immersion Phage-A reduced CFU by 1.37 logs on mustard seeds. Cocktail resulted in a 1.5-log reduction in CFU in the soaking water of broccoli seeds [77]
2005 Meat (broiler, turkey) PHL4 Sprayed Phage treatments reduced frequency of Salmonella recovery as compared with controls [78]
2008 Meat (raw/cooked beef) P7 Applied on top Reduction in CFU of 2-3 log at 5°C and approx. 6 log at 24°C [16]
2009 Fresh produce (tomatoes) Phage cocktail Phage + E. asburiae JX1 added to food Prevalence reduction of internalized S. Javiana, although the major suppressing effect was via antagonistic activity of E. asburiae JX1 [79]
2010 Fresh produce (mung bean sprouts and alfalfa Phage cocktail Phage + E. asburiae JX1 added to foods Combined biocontrol with E. asburiae and phage suppressed pathogen growth on mung beans and alfalfa seeds [80]
seeds)
2011 Meat (pig skin) Phage cocktail (PC1) Applied on top Above 99% reduction in CFU for MOI of 10 or above at 4°C for 96h [59]
2012 Ready-to-eat foods and chocolate milk FO1-E2 Added to foods and mixed in milk At 8°C no viable cells. At 15° C reduction of CFU by 5 logs on turkey deli meats and in chocolate milk and by 3 logs on hot dogs
Product
Phage(s)
Strategy
Main outcome
2005 2009
2010 2010 2011
Meat (ground beef)
Fresh produce (melons, apples)
Fresh produce (honeydew melon)
Processed food (red-smear soft cheese)
Processed food (cooked ham) Fresh produce (ready-to-eat products) Meat
(salmon fillet) Meat
(catfish fillets) Processed food (red-smear cheese) Processed food (ready-to-eat chicken)
Phage-nisin mixture Phage cocktail (LM-103, LM-102) combined with
Phage cocktail
P100 P100
A511, P100
P100 P100 A511
FWLLM1
Applied on top
Applied on top or sprayed
Sprayed
Applied to surfaces during the rind washings
Applied on top
Added to foods
Applied on top Applied on top Applied on top
Added to foods
Phage-nisin mixture was effective in broth but not in buffer or on raw beef
Phage caused a CFU reduction of 2.0 to 4.6 log in melons and only 0.4 log in apples. Phage + nisin reduced CFU by 5.7 (melon) and 2.3 (apple) log
Spraying melon pieces 0 h up to 1 h after Listeria challenge reduced CFU by 6.8 log units after 7 days of storage
Reduction of CFU or complete eradication during the rind washings
Rapid 1 log reduction of CFU. 2 log reduction after 14 to 28 days of storage
In liquid foods, eradication ofbacterial cells. On solid foods reduction of CFU by up to 5 log
Complete inhibition of growth at 4°C for 12 days, at
10°C for 8 days, and at 30°C for 4 days
Reduction of CFU by 1.4-2.0 log units at 4°C, 1.7-2.1
logs at 10°C, and 1.6-2.3 logs at 22°C
CFU counts dropped 3 logs after 22 days. Repeated
application of A511 further delayed re-growth
Reduction of CFU by 2.5 log at 30°C. At 5°C, regrowth was prevented over 21 days
[81] [82]
due to the human handling of the food products [93]. S. aureus can cause toxin-mediated diseases within 1 to 6 h after consumption of contaminated foods; nevertheless the symptoms are usually mild and most people recover within 1-3 days [94]. The annual estimated loss worldwide due to mastitis in adult dairy cows is of 35 billion US dollars [95]. Phage research has focused on the treatment of mastitis in lactating dairy cows and mostly in dairy food products (Table 5).
The experimental work of phage therapy on dairy cattle to control S. aureus on teats shows that, although there was no increase in somatic cell counts in milk samples indicating that the phages did not irritate the animal [96, 97], there were no statistically different outcomes between phage-treated and placebo groups [97]. This suggests that the use of phage as teat washes or sanitizers will not reduce the incidence of S. aureus mostly due to the barriers to phage-mediated bacteria lysis which are present in the bovine mammary gland. The suggested reasons for the inefficacy of phage K are the degradation or inactivation of the phage particles within the gland and the inhibitory effects of raw milk [97]. Also, it has been reported that milk whey prevents phages from reaching their host cell surface [98] due to the agglutination of S. aureus cells upon contact with raw whey [98, 99]. However, the work by Garcia et al. [100] on the use of phages in curd manufacturing processes provides evidence that phages are stable and active during enzymatic curd formation suggesting that pH is, in fact, the most crucial inactivation factor. Furthermore, these authors postulated
that the activity in milk of the phages used in their work was due to their dairy origin.
Postharvest applications in pasteurized milk show that the use of combined phage treatments with nisin and high hydrostatic pressure could synergistically be used to reduce staphylococcal contamination compared to each treatment alone [101, 102]. Nevertheless, care should be taken in regard to combined phage-nisin application since nisin-adapted strains can seriously compromise phage activity [101]. Inactivation of S. aureus has also been accomplished in both fresh and hard-type cheese using a phage cocktail during cheese manufacturing [103]. While for fresh cheese staphylococcal cells could not be detectable until the end of the curdling process (after 24 h), in hard cheeses, the presence of staphylococcal strains continuously dropped to 1.24 log CFU per gram atthe endofripening [103] providing evidence that phages can be successfully used to control S. aureus in dairy food products.
Today, there are phage-based diagnostic tools available to detect S. aureus, including antibiotic resistant (MRSA) and susceptible (MSSA) strains (Microphage, Inc.).
Besides the five foodborne pathogens described above, several other foodborne pathogens are responsible for illnesses, hospitalizations, and deaths, such as Clostridium spp., Shigella spp., and Vibrio spp. Nonetheless, these are not discussed further as to date there is a limited number of articles on the use of phage against these pathogens.
Year Animal/product Phage(s) Strategy Main outcome Refs
Preharvest application
2005 Ruminant (dairy cattle) K, CS1, DW2 Syringe applied into the teat sinus No detectable increase in somatic cell counts in milk [96]
2006 Ruminant (lactating dairy cattle) K Intramammary infusions Cure rate comparable in phage-treated and saline-treated quarters. No large increase of somatic cell count in the milk when phage was infused into quarters [97]
with S. aureus infection
Postharvest application
Raw and
2005 ready-to-eat foods (milk products and derivatives) K Added to milk Phages adsorption was reduced in raw milk and replication inhibited [99]
2006 Raw food K Added to raw milk Phage attachment and lysis inhibition due to [98]
(raw milk whey) whey adsorption of whey proteins to the S. aureus cell. S. aureus not detectable after 4 h at 25° C in acid curd
2007 Processes food (milk curd) Cocktail ($88 and $35) Added to pasteurized whole milk and total clearance within 1 h of incubation at 30° C in renneted curd. The addition of the phage cocktail to milk prior to acid and enzymatic curd manufacture eliminates S. aureus by up to 6 log units [100]
2008 Ready-to-eat foods (pasteurized milk) Cocktail ($35, $88) with nisin Cocktail Applied to foods and mixed in milk Nisin-phages application decreased S. aureus by 1 log unit more than in each antimicrobial agent alone (24 h at 37°C). Nisin-resistant phenotypes acquired resistance to phage, but phage resistance did not necessarily confer nisin resistance [101]
2012 Ready-to-eat foods (pasteurized milk) (philPLA35, philPLA88 with high hydrostatic pressure (HHP) Applied to foods and added to milk Combination of HHP and phage resulted in S. aureus elimination within the 48 h regardless of the initial contamination level (1 X 106 or 1 X 104 CFU per mL) [102]
vB_SauS-phi- Added to pasteurized milk vat Phage cocktail led to undetectable limits ofS. aureus
2012 Ready-to-eat foods IPLA35, after 6 h in fresh cheese and continuous reductions in [103]
(cheese) vB_SauS-phi-SauS- hard cheese. In curd a reduction of 4.64 log CFU per g
IPLA88 was obtained compared with control
5. Concluding Remarks and Future Perspectives
Soon after the discovery of phages, several studies produced negative results frequently associated with their inappropriate usage, including administration to treat viral and unknown agent diseases. Now, when once more the potential of phages is being evaluated mostly due to the increasing emergence of multiresistant strains, it is important not to repeat the same mistakes. Furthermore, the adoption of phage administration and delivery routes, control procedures, concentrations, and timings of application among other parameters based on results found in the literature must be done with caution as the efficacy of each phage or cocktail of phages is highly dependent on the phage-host systems in study. Furthermore, sampling of experimentally contaminated animals and foods and their treatment/control with phage should mimic as much as possible the conditions found at preharvest and postharvest (slaughterhouse, processing, and at retail) stages. For example, to estimate the input of the slaughterhouse into the process chain, samples should be collected before and after cooling. Ready-to-eat food usually undergoes several processing stages, and thus
it may be reasonable to verify the efficacy of phage after the process where phages were applied or, at least, at the end of the processing stage. Nevertheless, it is well evident that even after phage treatment, changes in food items still occur during transport and storage. Future studies should also focus on improving the general understanding of the mechanisms of phage resistance acquired by the hosts and the rate of elimination from the animal body. In addition, further evaluation of phage for biosanitation purposes is still not well documented for the pathogens listed above.
Although the results of the above-described studies appear to be encouraging, they should be interpreted cautiously. For instance, some phage studies have proven that phages are inefficient in reducing their host, such as phage delivery on foods and drinking water for controlling E. coli 0157: H7 in poultry and ruminants, and reduced efficacy of some of the phages reported. Also, the emergence of phage-resistant phenotypes is reported by several authors; however this has not significantly affected the results of the phage trials on animals and can be managed with using phages which target those resistant phenotypes. Nonetheless, summarizing briefly the results described recently: (i) phage therapy is
able to reduce foodborne pathogen levels in animals and consequently control the pathogen load on entry at the slaughterhouses; (ii) the strategies applied for phage biocon-trol of pathogens in foods reduce significantly the levels in a variety of products and seem to be a promising alternative to traditional food safety and preservation measures; (iii) phage use in agricultural settings is as efficient or more than the conventionally used agents to control the growth of plant-based bacterial pathogens. However, the future of phages in food safety is further dependent on the regulatory agencies that still display uneasiness with using phages, mostly due to a scarcity of strong scientific evidence generated through fully controlled clinical trials under the supervision of ethical committees and in compliance with the highest regulatory standards of leading Western jurisdictions [90]. Also, there is a need to educate farmers, producers, and the general public about the advantages of their use.
Authors' Contribution
S. M. Sillankorva and H. Oliveira equally contributed to the paper.
Acknowledgments
This work was supported by a Grant from the Portuguese Foundation for Science and Technology in the scope of the Projects PTDC/AGR-ALI/100492/2008. H. Oliveira and S. M. Sillankorva acknowledge the FCT Grants SFRH/BD/ 63734/2009 and SFRH/BPD/48803/2008, respectively.
References
[1] CDC, CDC Estimates ofFoodborne Illness in the United States, Center for Disease Control and Prevention (CDC), 2011.
[2] J. Rocourt, G. Moy, K. Vierk, and J. Schlundt, The Present State of Foodborne Disease in OECD Countries, 2003.
[3] J. Maukonen, J. Matto, G. Wirtanen, L. Raaska, T. MattilaSandholm, and M. Saarela, "Methodologies for the characterization of microbes in industrial environments: a review," Journal ofIndustrial Microbiology and Biotechnology, vol. 30, no. 6, pp. 327-356, 2003.
[4] J. T. Holah, J. H. Taylor, D. J. Dawson, and K. E. Hall, "Biocide use in the food industry and the disinfectant resistance of persistent strains of Listeria monocytogenes and Escherichia coli," Journal of Applied Microbiology, vol. 92, pp. 111S-120S, 2002.
[5] K. Lewis, "Multidrug tolerance of biofilms and persister cells," Current Topics in Microbiology and Immunology, vol. 322, pp. 107-131,2008.
[6] R. J. Atterbury, P. L. Connerton, C. E. R. Dodd, C. E. D. Rees, and I. F. Connerton, "Application of host-specific bacteriophages to the surface of chicken skin leads to a reduction in recovery of Campylobacter jejuni," Applied and Environmental Microbiology, vol. 69, no. 10, pp. 6302-6306, 2003.
[7] F. C. Hsu, Y. S. C. Shieh, and M. D. Sobsey, "Enteric bacteriophages as potential fecal indicators in ground beef and poultry meat," Journal of Food Protection, vol. 65, no. 1, pp. 93-99, 2002.
[8] J. E. Kennedy, C. I. Wei, and J. L. Oblinger, "Distribution of coliphages in various foods," Journal of Food Protection, vol. 49, no. 12, pp. 944-951, 1986.
[9] V. B. Suarez, A. Quiberoni, A. G. Binetti, and J. A. Rein-heimer, "Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries," Journal of Food Protection, vol. 65, no. 10, pp. 1597-1604, 2002.
[10] D. L. Croci, D. de Medici, C. Scalfaro et al., "Determination of enteroviruses, hepatitis A virus, bacteriophages and Escherichia coli in Adriatic Sea mussels," Journal of Applied Microbiology, vol. 88, no. 2, pp. 293-298, 2000.
[11] H. Rosenquist, N. L. Nielsen, H. M. Sommer, B. N0rrung, and B. B. Christensen, "Quantitative risk assessment of human campylobacteriosis associated with thermophilic Campylobacter species in chickens," International Journal of Food Microbiology, vol. 83, no. 1, pp. 87-103, 2003.
[12] L. Snyder and W. Champness, "Lytic bacteriophages: genetic analysis and use in transduction," in Molecular Genetics of Bacteria, L. S. A. W. Champness, Ed., pp. 293-305, ASM Press, Washington, DC, USA, 2007.
[13] D. Goode, V. M. Allen, and P. A. Barrow, "Reduction of experimental Salmonella and Campylobacter contamination of chicken skin by application of lytic bacteriophages," Applied and Environmental Microbiology, vol. 69, no. 8, pp. 5032-5036, 2003.
[14] G.G. Greer, "Psychrotrophic bacteriophages for beef spoilage pseudomonads," Journal of Food Protection, vol. 45, pp. 13181325, 1982.
[15] G. G. Greer, "Effect of phage concentration, bacterial density, and temperature on phage control of beef spoilage," Journal ofFood Science, vol. 53, pp. 1226-1227, 1988.
[16] T. Bigwood, J. A. Hudson, C. Billington, G. V. Carey-Smith, and J. A. Heinemann, "Phage inactivation of foodborne pathogens on cooked and raw meat," Food Microbiology, vol. 25, no. 2, pp. 400-406, 2008.
[17] E. R. S. E. Data, Foodborne Illness Cost Calculator: Salmonella, 2010.
[18] G. G. Greer, "Bacteriophage control of foodborne bacteria," Journal of Food Protection, vol. 68, no. 5, pp. 1102-1111,2005.
[19] J. B. Kaper, "Enterohemorrhagic Escherichia coli" Current Opinion in Microbiology, vol. 1, no. 1, pp. 103-108, 1998.
[20] J. B. Russell, F. Diez-Gonzalez, and G. N. Jarvis, "Symposium: farm health and safety invited review: effects of diet shifts on Escherichia coli in cattle," Journal of Dairy Science, vol. 83, no. 4, pp. 863-873, 2000.
[21] J. M. Whichard, N. Sriranganathan, and F. W. Pierson, "Suppression of Salmonella growth by wild-type and large-plaque variants of bacteriophage Felix O1 in liquid culture and on chicken frankfurters," Journal of Food Protection, vol. 66, no. 2, pp. 220-225, 2003.
[22] W. E. Huff, G. R. Huff, N. C. Rath, J. M. Balog, and A. M. Donoghue, "Prevention ofEscherichia coli infection in broiler chickens with a bacteriophage aerosol spray," Poultry Science, vol. 81, no. 10, pp. 1486-1491, 2002.
[23] W. E. Huff, G. R. Huff, N. C. Rath, J. M. Balog, and A. M. Donoghue, "Evaluation of aerosol spray and intramuscular injection of bacteriophage to treat an Escherichia coli respiratory infection," Poultry Science, vol. 82, no. 7, pp. 1108-1112, 2003.
[24] W. E. Huff, G. R. Huff, N. C. Rath et al., "Prevention of Escherichia coli respiratory infection in broiler chickens with bacteriophage (SPR02)," Poultry Science, vol. 81, no. 4, pp. 437-441, 2002.
[25] L. D. Goodridge and B. Bisha, "Phage-based biocontrol strategies to reduce foodborne pathogens in foods," Bacteriophage, vol. 1, no. 3, pp. 130-137, 2011.
[26] H. W. Smith, M. B. Huggins, and K. M. Shaw, "Factors influencing the survival and multiplication of bacteriophages in calves and in their environment," Journal of General Microbiology, vol. 133, no. 5, pp. 1127-1135, 1987.
[27] S. Hagens and M. J. Loessner, "Application ofbacteriophages for detection and control of foodborne pathogens," Applied Microbiology and Biotechnology, vol. 76, no. 3, pp. 513-519,
[28] K. Stanford, T. A. McAllister, Y. D. Niu et al., "Oral delivery systems for encapsulated bacteriophages targeted Escherichia coli o157: H7 in feedlot cattle," Journal of Food Protection, vol. 73, no. 7, pp. 1304-1312, 2010.
[29] S. J. Bach, T. A. McAllister, D. M. Veira, V. P. J. Gannon, and R. A. Holley, "Effect of bacteriophage DC22 on Escherichia coli O157:H7 in an artificial rumen system (Rusitec) and inoculated sheep," Animal Research, vol. 52, no. 2, pp. 89101,2003.
[30] W. E. Huff, G. R. Huff, N. C. Rath, and A. M. Donoghue, "Evaluation of the influence of bacteriophage titer on the treatment of colibacillosis in broiler chickens," Poultry Science, vol. 85, no. 8, pp. 1373-1377, 2006.
[31] R. R. Raya, P. Varey, R. A. Oot et al., "Isolation and characterization of a new T-even bacteriophage, CEV1, and determination of its potential to reduce Escherichia coli O157:H7 levels in sheep," Applied and Environmental Microbiology, vol. 72, no. 9, pp. 6405-6410, 2006.
[32] H. Sheng, H. J. Knecht, I. T. Kudva, and C. J. Hovde, "Application of bacteriophages to control intestinal Escherichia coli O157:H7 levels in ruminants," Applied and Environmental Microbiology, vol. 72, no. 8, pp. 5359-5366, 2006.
[33] E. A. Rozema, T. P. Stephens, S. J. Bach et al., "Oral and rectal administration of bacteriophages for control of Escherichia coli coli O157:H7 in feedlot cattle," Journal of Food Protection, vol. 72, no. 2, pp. 241-250, 2009.
[34] A. Oliveira, R. Sereno, and J. Azeredo, "In vivo efficiency evaluation of a phage cocktail in controlling severe colibacillosis in confined conditions and experimental poultry houses," Veterinary Microbiology, vol. 146, no. 3-4, pp. 303-308, 2010.
[35] L. Rivas, B. Coffey, O. McAuliffe et al., "In vivo and ex vivo evaluations of bacteriophages e11/2 and e4/1c for use in the control of Escherichia coli O157:H7," Applied and Environmental Microbiology, vol. 76, no. 21, pp. 7210-7216, 2010.
[36] R. R. Raya, R. A. Oot, B. Moore-Maley et al., "Naturally resident and exogenously applied T4-like and T5-like bacte-riophages can reduce Escherichia coli O157:H7 levels in sheep guts," Bacteriophage, vol. 1, no. 1, pp. 15-24, 2011.
[37] G. O'Flynn, R. P. Ross, G. F. Fitzgerald, and A. Coffey, "Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7," Applied and Environmental Microbiology, vol. 70, no. 6, pp. 3417-3424, 2004.
[38] T. Abuladze, M. Li, M. Y. Menetrez, T. Dean, A. Senecal, and A. Sulakvelidze, "Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7," Applied and Environmental Microbiology, vol. 74, no. 20, pp. 6230-6238,
[39] M. Sharma, J. R. Patel, W. S. Conway, S. Ferguson, and A. Sulakvelidze, "Effectiveness of bacteriophages in reducing Escherichia coli O157:H7 on fresh-cut cantaloupes and
lettuce," Journal of Food Protection, vol. 72, no. 7, pp. 14811485, 2009.
[40] S. Viazis, M. Akhtar, J. Feirtag, and F. Diez-Gonzalez, "Reduction of Escherichia coli O157:H7 viability on hard surfaces by treatment with a bacteriophage mixture," International Journal of Food Microbiology, vol. 145, no. 1, pp. 37-42, 2011.
[41] J. Patel, M. Sharma, P. Millner, T. Calaway, and M. Singh, "Inactivation of Escherichia coli O157:H7 attached to spinach harvester blade using bacteriophage," Foodborne Pathogens and Disease, vol. 8, no. 4, pp. 541-546, 2011.
[42] Y. D. Niu, Y. Xu, T. A. McAllister et al., "Comparison of fecal versus rectoanal mucosal swab sampling for detecting Escherichia coli O157:H7 in experimentally inoculated cattle used in assessing bacteriophage as a mitigation strategy," Journal of Food Protection, vol. 71, no. 4, pp. 691-698, 2008.
[43] J. E. Moore, D. Corcoran, J. S. Dooley et al., "Campylobacter," Veterinary Research, vol. 36, no. 3, pp. 351-382, 2005.
[44] T. Humphrey, S. O'Brien, and M. Madsen, "Campylobacters as zoonotic pathogens: a food production perspective," International Journal of Food Microbiology, vol. 117, no. 3, pp. 237-257, 2007.
[45] S. J. Evans and A. R. Sayers, "A longitudinal study of campylobacter infection of broiler flocks in Great Britain," Preventive Veterinary Medicine, vol. 46, no. 3, pp. 209-223, 2000.
[46] S. P. Oliver, B. M. Jayarao, and R. A. Almeida, "Foodborne pathogens in milk and the dairy farm environment: food safety and public health implications," Foodborne Pathogens and Disease, vol. 2, no. 2, pp. 115-129, 2005.
[47] F. Reich, V. Atanassova, E. Haunhorst, and G. Klein, "The effects of Campylobacter numbers in caeca on the contamination of broiler carcasses with Campylobacter," International Journal of Food Microbiology, vol. 127, no. 1-2, pp. 116-120, 2008.
[48] C. L. Carrillo, R. J. Atterbury, A. El-Shibiny et al., "Bacteriophage therapy to reduce Campylobacter jejuni colonization of broiler chickens," Applied and Environmental Microbiology, vol. 71, no. 11, pp. 6554-6563, 2005.
[49] A. El-Shibiny, A. Scott, A. Timms, Y. Metawea, P. Connerton, and I. Connerton, "Application of a group II campylobacter bacteriophage to reduce strains of Campylobacter jejuni and campylobacter coli colonizing broiler chickens," Journal of Food Protection, vol. 72, no. 4, pp. 733-740, 2009.
[50] C. M. Carvalho, B. W. Gannon, D. E. Halfhide et al., "The in vivo efficacy of two administration routes of a phage cocktail to reduce numbers ofCampylobacter coli and Campylobacter jejuni in chickens," BMC Microbiology, vol. 10, article 232, 2010.
[51] A. E. Scott, A. R. Timms, P. L. Connerton, C. Loc Carrillo, K. Adzfa Radzum, and I. F. Connerton, "Genome dynamics of Campylobacter jejuni in response to bacteriophage predation," PLoS pathogens, vol. 3, no. 8, p. e119, 2007.
[52] T. Ica, V. Caner, O. Istanbullu et al., "Characterization of mono- and mixed-culture Campylobacter jejuni biofilms," Applied and Environmental Microbiology, vol. 78, no. 4, pp. 1033-1038, 2012.
[53] P. Siringan, P. L. Connerton, R. J. H. Payne, and I. F. Connerton, "Bacteriophage-mediated dispersal of Campylobacter jejuni biofilms," Applied and Environmental Microbiology, vol. 77, no. 10, pp. 3320-3326, 2011.
[54] F. I. F. R. A. B. O. Germany, The Return of the Germs, 2004.
[55] EFSA, "The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne
outbreaks in 2009," The European Food Safety Authority Journal, vol. 9, no. 3, p. 2090, 2011.
[56] G. Barbara, V. Stanghellini, C. Berti-Ceroni et al., "Role of antibiotic therapy on long-term germ excretion in faeces and digestive symptoms after Salmonella infection," Alimentary Pharmacology and Therapeutics, vol. 14, no. 9, pp. 1127-1131, 2000.
[57] Y. Ma, J. C. Pacan, Q. Wang et al., "Microencapsulation of bacteriophage felix o1 into chitosan-alginate microspheres for oral delivery," Applied and Environmental Microbiology, vol. 74, no. 15, pp. 4799-4805, 2008.
[58] J. A. Wagenaar, M. A. P. V. Bergen, M. A. Mueller, T. M. Wassenaar, and R. M. Carlton, "Phage therapy reduces Campylobacter jejuni colonization in broilers," Veterinary Microbiology, vol. 109, no. 3-4, pp. 275-283, 2005.
[59] S. Guenther, O. Herzig, L. Fieseler, J. Klumpp, and M. J. Loessner, "Biocontrol of Salmonella Typhimurium in RTE foods with the virulent bacteriophage FO1-E2," International Journal of Food Microbiology, vol. 154, no. 1-2, pp. 66-72, 2012.
[60] H. P. R. Seeliger and F. Jones, "Listeria," in Bergey's Manual of Systematic Bacteriology, B. Williams and M. D. Wilkins, Eds., pp. 1235-1245, 1986.
[61] Y. C. Chan and M. Wiedmann, "Physiology and genetics of Listeria monocytogenes survival and growth at cold temperatures," Critical Reviews in Food Science and Nutrition, vol. 49, no. 3, pp. 237-253, 2009.
[62] R. B. Tompkin, "Control of Listeria monocytogenes in the food-processing environment," Journal of Food Protection, vol. 65, no. 4, pp. 709-725, 2002.
[63] H. de Valk, C. Jacquet, V. Goulet et al., "Surveillance oflisteria infections in Europe," Euro Surveillance, vol. 10, no. 10, pp. 251-255,2005.
[64] K. A. Soni and R. Nannapaneni, "Removal of Listeria monocytogenes biofilms with bacteriophage P100," Journal of Food Protection, vol. 73, no. 8, pp. 1519-1524, 2010.
[65] I. B. Sklar and R. D. Joerger, "Attempts to utilize bacte-riophage to combat salmonella enterica serovar enteritidis infection in chickens," Journal of Food Safety, vol. 21, no. 1, pp. 15-29,2001.
[66] N. Lee and D. L. Harris, "The effect of bacteriophage treatment as a preharvest intervention strategy to reduce the rapid dissemination of Salmonella typhimurium in pigs," in Proceedings of the American Association of Swine Veterinarians, pp. 555-557, 2001.
[67] L. Fiorentin, N. D. Vieira, and W. Barioni Jr., "Oral treatment with bacteriophages reduces the concentration of Salmonella Enteritidis PT4 in caecal contents of broilers," Avian Pathology, vol. 34, no. 3, pp. 258-263, 2005.
[68] H. Toro, S. B. Price, S. McKee et al., "Use of bacterio-phages in combination with competitive exclusion to reduce Salmonella from infected chickens," Avian Diseases, vol. 49, no. 1,pp. 118-124, 2005.
[69] R. J. Atterbury, M. A. P. van Bergen, F. Ortiz et al., "Bacteriophage therapy to reduce Salmonella colonization of broiler chickens," Applied and Environmental Microbiology, vol. 73, no. 14, pp. 4543-4549, 2007.
[70] R. L. Andreatti Filho, J. P. Higgins, S. E. Higgins et al., "Ability of bacteriophages isolated from different sources to reduce Salmonella enterica serovar Enteritidis in vitro and in vivo," Poultry Science, vol. 86, no. 9, pp. 1904-1909, 2007.
[71] C. Borie, I. Albala, P. Sanchez et al., "Bacteriophage treatment reduces Salmonella colonization of infected chickens," Avian Diseases, vol. 52, no. 1, pp. 64-67, 2008.
[72] S. K. Wall, J. Zhang, M. H. Rostagno, and P. D. Ebner, "Phage therapy to reduce preprocessing Salmonella infections in market-weight swine," Applied and Environmental Microbiology, vol. 76, no. 1, pp. 48-53, 2010.
[73] T. R. Callaway, R. C. Anderson, T. S. Edrington et al., "What are we doing about Escherichia coli O157:H7 in cattle?" Journal ofAnimal Science, vol. 82, pp. E93-E99, 2004.
[74] T.-H. Lim, D.-H. Lee, Y.-N. Lee et al., "Efficacy of bacte-riophage therapy on horizontal transmission of Salmonella Gallinarum on commercial layer chickens," Avian Diseases, vol. 55, no. 3, pp. 435-438, 2011.
[75] R. Modi, Y. Hirvi, A. Hill, and M. W. Griffiths, "Effect of phage on survival of Salmonella Enteritidis during manufacture and storage of Cheddar cheese made from raw and pasteurized milk," Journal of Food Protection, vol. 64, no. 7, pp. 927-933, 2001.
[76] B. Leverentz, W. S. Conway, Z. Alavidze et al., "Examination of bacteriophage as a biocontrol method for Salmonella on fresh-cut fruit: a model study," Journal of Food Protection, vol. 64, no. 8, pp. 1116-1121,2001.
[77] S. Pao, S. P. Randolph, E. W. Westbrook, and H. Shen, "Use of bacteriophages to control Salmonella in experimentally contaminated sprout seeds," Journal of Food Science, vol. 69, no. 5, pp. M127-M130, 2004.
[78] J. P. Higgins, S. E. Higgins, K. L. Guenther et al., "Use of a specific bacteriophage treatment to reduce Salmonella in poultry products," Poultry Science, vol. 84, no. 7, pp. 1141— 1145, 2005.
[79] J. Ye, M. Kostrzynska, K. Dunfield, and K. Warriner, "Evaluation of a biocontrol preparation consisting of enterobacter asburiae JX1 and a lytic bacteriophage cocktail to suppress the growth of salmonella javiana associated with tomatoes," Journal of Food Protection, vol. 72, no. 11, pp. 2284-2292, 2009.
[80] S. P. T. Hooton, R. J. Atterbury, and I. F. Connerton, "Application ofa bacteriophage cocktail to reduce Salmonella Typhimurium U288 contamination on pig skin," International Journal of Food Microbiology, vol. 151, no. 2, pp. 157163,2011.
[81] G. A. Dykes and S. M. Moorhead, "Combined antimicrobial effect of nisin and a listeriophage against Listeria monocytogenes in broth but not in buffer or on raw beef" International Journal of Food Microbiology, vol. 73, no. 1, pp. 71-81, 2002.
[82] B. Leverentz, W. S. Conway, M. J. Camp et al., "Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin," Applied and Environmental Microbiology, vol. 69, no. 8, pp. 4519-4526, 2003.
[83] B. Leverentz, W. S. Conway, W. Janisiewicz, and M. J. Camp, "Optimizing concentration and timing of a phage spray application to reduce Listeria monocytogenes on honeydew melon tissue," Journal of Food Protection, vol. 67, no. 8, pp. 1682-1686, 2004.
[84] R. M. Carlton, W. H. Noordman, B. Biswas, E. D. de Meester, andM. J. Loessner, "Bacteriophage P100 for control of Listeria monocytogenes in foods: genome sequence, bioinformatic analyses, oral toxicity study, and application," Regulatory Toxicology and Pharmacology, vol. 43, no. 3, pp. 301-312, 2005.
[85] A. Holck and J. Berg, "Inhibition of Listeria monocytogenes in cooked ham by virulent bacteriophages and protective cultures," Applied and Environmental Microbiology, vol. 75, no. 21, pp. 6944-6946, 2009.
[86] S. Guenther, D. Huwyler, S. Richard, and M. J. Loessner, "Virulent bacteriophage for efficient biocontrol of Listeria monocytogenes in ready-to-eat foods," Applied and Environmental Microbiology, vol. 75, no. 1, pp. 93-100, 2009.
[87] K. A. Soni, R. Nannapaneni, and S. Hagens, "Reduction of Listeria monocytogenes on the surface of fresh channel catfish fillets by bacteriophage listex p100," Foodborne Pathogens and Disease, vol. 7, no. 4, pp. 427-434, 2010.
[88] S. Guenther and M. J. Loessner, "Bacteriophage biocontrol of Listeria monocytogenes on soft ripened white mold and red-smear cheeses," Bacteriophage, vol. 1, no. 2, pp. 94-100, 2011.
[89] B. Bigot, W. J. Lee, L. McIntyre et al., "Control of Listeria monocytogenes growth in a ready-to-eat poultry product using a bacteriophage," Food Microbiology, vol. 28, no. 8, pp. 1448-1452, 2011.
[90] EFSA, "The use and mode of action of bacteriophages in food production. Scientific opinion of the panel on biological hazards," The European Food Safety Authority Journal, vol. 1076, pp. 1-26, 2009.
[91] Y. Le Loir, F. Baron, and M. Gautier, "Staphylococcus aureus and food poisoning," Genetics and Molecular Research, vol. 2, no. 1, pp. 63-76, 2003.
[92] M. A. Argudin, M. C. Mendoza, and M. R. Rodicio, "Food poisoning and Staphylococcus aureus enterotoxins," Toxins, vol. 2, no. 7, pp. 1751-1773, 2010.
[93] V. Spanu, C. Spanu, S. Virdis, F. Cossu, C. Scarano, and E. P. L. de Santis, "Virulence factors and genetic variability of Staphylococcus aureus strains isolated from raw sheep's milk cheese," International Journal of Food Microbiology, vol. 153, no. 1-2, pp. 53-57, 2012.
[94] "What You Need to Know about Foodborne Illness-Causing Organisms in the U.S," U.S. Food and Drug Administratio, 2012.
[95] G. J. Wellenberg, W. H. M. van der Poel, and J. T. van Oirschot, "Viral infections and bovine mastitis: a review," Veterinary Microbiology, vol. 88, no. 1, pp. 27-45, 2002.
[96] S. O'Flaherty, R. P. Ross, J. Flynn, W. J. Meaney, G. F. Fitzgerald, and A. Coffey, "Isolation and characterization of two anti-staphylococcal bacteriophages specific for pathogenic Staphylococcus aureus associated with bovine infections," Letters in Applied Microbiology, vol. 41, no. 6, pp. 482-486, 2005.
[97] J. J. Gill, J. C. Pacan, M. E. Carson, K. E. Leslie, M. W. Griffiths, and P. M. Sabour, "Efficacy and pharmacokinet-ics of bacteriophage therapy in treatment of subclinical Staphylococcus aureus mastitis in lactating dairy cattle," Antimicrobial Agents and Chemotherapy, vol. 50, no. 9, pp. 2912-2918, 2006.
[98] J. J. Gill, P. M. Sabour, K. E. Leslie, and M. W. Griffiths, "Bovine whey proteins inhibit the interaction of Staphylococcus aureus and bacteriophage K," Journal of Applied Microbiology, vol. 101, no. 2, pp. 377-386, 2006.
[99] S. O'Flaherty, A. Coffey, W. J. Meaney, G. F. Fitzgerald, and R. P. Ross, "Inhibition of bacteriophage K proliferation on Staphylococcus aureus in raw bovine milk," Letters in Applied Microbiology, vol. 41, no. 3, pp. 274-279, 2005.
[100] P. Garcia, C. Madera, B. Martinez, and A. Rodriguez, "Biocontrol of Staphylococcus aureus in curd manufacturing processes using bacteriophages," International Dairy Journal, vol. 17, no. 10, pp. 1232-1239, 2007.
[101] B. Martinez, J. M. Obeso, A. Rodriguez, and P. Garcia, "Nisin-bacteriophage crossresistance in Staphylococcus aureus," International Journal ofFood Microbiology, vol. 122, no. 3, pp. 253-258, 2008.
[102] R. Tabla, B. Martinez, J. E. Rebollo et al., "Bacteriophage performance against Staphylococcus aureus in milk is improved by high hydrostatic pressure treatments," International Journal of Food Microbiology, vol. 156, no. 3, pp. 209-213, 2012.
[103] E. Bueno, P. Garcia, B. Martinez, and A. Rodriguez, "Phage inactivation of Staphylococcus aureus in fresh and hard-type cheeses," International Journal of Food Microbiology, vol. 158, no. 1,pp. 23-27, 2012.
Copyright of International Journal of Microbiology is the property of Hindawi Publishing Corporation and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
