Natural Killers: Opportunities and Challenges for the Use of Bacteriophages in Microbial Food Safety from the One Health Perspective

Ingestion of food or water contaminated with pathogenic bacteria may cause serious diseases. The One Health approach may help to ensure food safety by anticipating, preventing, detecting, and controlling diseases that spread between animals, humans, and the environment. This concept pays special attention to the increasing spread and dissemination of antibiotic-resistant bacteria, which are considered one of the most important environment-related human and animal health hazards. In this context, the development of innovative, versatile, and effective alternatives to control bacterial infections in order to assure comprehensive food microbial safety is becoming an urgent issue. Bacteriophages (phages), viruses of bacteria, have gained significance in the last years due to the request for new effective antimicrobials for the treatment of bacterial diseases, along with many other applications, including biotechnology and food safety. This manuscript reviews the application of phages in order to prevent food- and water-borne diseases from a One Health perspective. Regarding the necessary decrease in the use of antibiotics, results taken from the literature indicate that phages are also promising tools to help to address this issue. To assist future phage-based real applications, the pending issues and main challenges to be addressed shortly by future studies are also taken into account.


Introduction
Food and water are the main routes of transmission of more than 200 known infectious diseases, many of which are attributed to bacteria [1]. Among these, the main bacterial foodborne pathogens, in terms of occurrence and seriousness, are Salmonella enterica, Campylobacter spp., Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Clostridium botulinum. Considering waterborne diseases, the genus Vibrio also enters this important group, besides E. coli and S. enterica serovar Typhi [2]. Besides the potential serious clinical symptoms, food-and water-borne diseases represent an enormous economic burden for health systems (treatment, potential hospitalization, loss of working days, etc.) and some of them, such us L. monocytogenes, can be fatal to humans. Accordingly, measures for the prevention of their presence and proliferation in food products should be comprehensive and strict.
On the other hand, food production is a complex and multifaceted procedure which starts from the growth of animals and the harvest of plants through different practices up to the point of their consumption by customer. Along this path, there are many chances for bacterial contamination. Many of those pathogenic bacteria are considered as ubiquitous and normal microbiota in the environment and in animals. They may cause infections as Considering the emergence of multidrug-resistant (MDR) bacteria, the development of innovative and effective alternatives to control bacterial infections is becoming an urgent issue. In this scenario, bacteriophages or phages, which are viruses that infect bacteria and the most abundant and diverse biological entities worldwide, are currently gaining an important prominence in the request of new effective alternatives to treat bacterial diseases in humans, animals, and plants [9,10]. Moreover, this "phage biocontrol", targeting specific pathogenic bacteria, is also increasingly accepted as a natural, effective, and inexpensive food (and feed) safety strategy [11,12].
Phages are usually classified according to the strategies they use to escape their hosts, into lysogenic (temperate) phages and lytic (virulent) phages [13,14]. Lytic phages cause the death (lysis) of their host at the end of their lytic cycle ( Figure 2) and, consequently, these are the most suitable phages to be used in biocontrol applications [15]. Because of their high specificity, phages eliminate target bacteria without affecting the normal and beneficial microbiota of the host, the food, or the environment. Moreover, as they are already widely present in the environment, phages are not supposed to have any harmful consequence on Considering the emergence of multidrug-resistant (MDR) bacteria, the development of innovative and effective alternatives to control bacterial infections is becoming an urgent issue. In this scenario, bacteriophages or phages, which are viruses that infect bacteria and the most abundant and diverse biological entities worldwide, are currently gaining an important prominence in the request of new effective alternatives to treat bacterial diseases in humans, animals, and plants [9,10]. Moreover, this "phage biocontrol", targeting specific pathogenic bacteria, is also increasingly accepted as a natural, effective, and inexpensive food (and feed) safety strategy [11,12].
Phages are usually classified according to the strategies they use to escape their hosts, into lysogenic (temperate) phages and lytic (virulent) phages [13,14]. Lytic phages cause the death (lysis) of their host at the end of their lytic cycle ( Figure 2) and, consequently, these are the most suitable phages to be used in biocontrol applications [15]. Because of their high specificity, phages eliminate target bacteria without affecting the normal and beneficial microbiota of the host, the food, or the environment. Moreover, as they are already widely present in the environment, phages are not supposed to have any harmful consequence on the quality of food or animal and human health [16]. The safety and effectiveness of phage-based biocontrollers is reflected in the fact that several preparations have been approved for use in food [17]. In addition, they can be used alone or in combination with other phages (phage cocktails) in order to achieve a broader host range, or with antibiotics or disinfectants to control bacterial infections more effectively [18]. In fact, phage-antibiotic synergy (knowns as the PAS effect), has been observed in some phageantibiotic combinations [19].
Foods 2023, 12,552 3 of 24 the quality of food or animal and human health [16]. The safety and effectiveness of phagebased biocontrollers is reflected in the fact that several preparations have been approved for use in food [17]. In addition, they can be used alone or in combination with other phages (phage cocktails) in order to achieve a broader host range, or with antibiotics or disinfectants to control bacterial infections more effectively [18]. In fact, phage-antibiotic synergy (knowns as the PAS effect), has been observed in some phage-antibiotic combinations [19]. Phages are potent antimicrobial agents against most pathogenic bacteria, and can be usefully implemented for their environmentally-friendly application at each stage of the farm-to-fork continuum. This corresponds to the One Health concept for reducing foodborne diseases: they can be used in many applications, such as phage-therapy in animal production, cleaning of the livestock, disinfection and sanitation of equipment and contact surfaces on farms and in industry, biocontrols in fresh meats and produce, and also as natural preservatives to extend product shelf-life [1,21,22].
Nevertheless, many factors, such as the target bacteria, the application route, the phage administration timing (prophylactic vs. therapeutic), the number of phage administrations (single vs. repeated), the number of phages used (single vs. cocktail), food composition, or the storage temperature, for instance, are factors to be taken into account, as they may imply differences in the results regarding the effects of phages on the biocontrol of pathogens in foods and animals [15,23].

Phage Biocontrol in Animal Husbandry for Food Production
The raise of MDR bacteria causing dissemination of AMR requires alternative strategies to combat pathogenic bacteria. Phages possess some unique characteristics, such as high species-specific nature, relatively easy handling, self-replication, and self-limiting [24,25], that make them a promising alternative for effectively inhibiting the colonization of pathogenic bacteria and reducing animal and zoonotic diseases [17,26,27].

Control of Campylobacter and Salmonella in Broilers
Campylobacter and Salmonella are widely distributed in most warm-blooded animals, such as poultry, and contaminate foods during slaughter, handling, and/or carcass processing. The main source of human infection for both bacteria is the consumption of contaminated products of animal origin, mainly undercooked eggs and poultry meat [28]. Due to their importance in public health, both bacteria are controlled by the European Community legislation. However, despite efforts to control them, new foodborne outbreaks (FBO) of salmonellosis and campylobacteriosis emerge every year [28,29].
Campylobacter is the most common foodborne pathogen causing zoonotic illnesses in humans. The majority of campylobacteriosis cases are caused by Campylobacter jejuni Phages are potent antimicrobial agents against most pathogenic bacteria, and can be usefully implemented for their environmentally-friendly application at each stage of the farm-to-fork continuum. This corresponds to the One Health concept for reducing foodborne diseases: they can be used in many applications, such as phage-therapy in animal production, cleaning of the livestock, disinfection and sanitation of equipment and contact surfaces on farms and in industry, biocontrols in fresh meats and produce, and also as natural preservatives to extend product shelf-life [1,21,22].
Nevertheless, many factors, such as the target bacteria, the application route, the phage administration timing (prophylactic vs. therapeutic), the number of phage administrations (single vs. repeated), the number of phages used (single vs. cocktail), food composition, or the storage temperature, for instance, are factors to be taken into account, as they may imply differences in the results regarding the effects of phages on the biocontrol of pathogens in foods and animals [15,23].

Phage Biocontrol in Animal Husbandry for Food Production
The raise of MDR bacteria causing dissemination of AMR requires alternative strategies to combat pathogenic bacteria. Phages possess some unique characteristics, such as high species-specific nature, relatively easy handling, self-replication, and self-limiting [24,25], that make them a promising alternative for effectively inhibiting the colonization of pathogenic bacteria and reducing animal and zoonotic diseases [17,26,27].

Control of Campylobacter and Salmonella in Broilers
Campylobacter and Salmonella are widely distributed in most warm-blooded animals, such as poultry, and contaminate foods during slaughter, handling, and/or carcass processing. The main source of human infection for both bacteria is the consumption of contaminated products of animal origin, mainly undercooked eggs and poultry meat [28]. Due to their importance in public health, both bacteria are controlled by the European Community legislation. However, despite efforts to control them, new foodborne outbreaks (FBO) of salmonellosis and campylobacteriosis emerge every year [28,29].
Campylobacter is the most common foodborne pathogen causing zoonotic illnesses in humans. The majority of campylobacteriosis cases are caused by Campylobacter jejuni (∼89%) and Campylobacter coli (∼10%), with only a few cases (<1%) associated with C. fetus, C. upsaliensis, and C. lari [28]. Although treatment is not generally required, quinolones, macrolides, and tetracyclines are antibiotics used to combat severe infections [30,31]. The alarming emergence of Campylobacter resistance to these drugs compromises the effectiveness of therapeutic treatments and lead the World Health Organization (WHO) to include Campylobacter in its global priority list of antibiotic-resistant pathogens [32].
Poultry is considered the natural primary reservoir of Campylobacter spp., with C. jejuni being the predominant species colonizing broiler chickens. Colonization naturally occurs by horizontal transmission from the environment, and the infection rapidly spreads within the flock, reaching more than 10 7 CFU/g in their intestinal tract before slaughter [33]. The prevention and control of Campylobacter in poultry is a food safety issue of high priority, since it is widely accepted as a significant risk factor of human campylobacteriosis. Reducing the Campylobacter load in broiler intestines by 3 logs prior to slaughter was estimated to reduce the risk of human campylobacteriosis attributable to the consumption of poultry meat by 58% [34].
Even though no phage-based products with specific activity against Campylobacter are commercially available yet, phage biocontrol is one of the most promising alternatives under development to address the reduction in this pathogen in the poultry reservoir [35]. Campylobacter-infecting phages (also called campylophages) have been isolated wherever their hosts are present, including in both poultry environmental samples and food products [33,36]. Campylophages have been classified into three groups (groups I, II, and III) according to their genome size [37]. Group I phages (320 kb) have rarely been isolated, whereas phages of group II (180 kb; Cp220virus) and group III (140 kb; Cp8virus) are common and contact their target hosts via flagella and capsule polysaccharide (CPS) receptors, respectively [33].
Different studies have reported the use of campylophages to reduce Campylobacter counts in the gastrointestinal tracts of broiler chickens (Table 1), without affecting their health and well-being [38] or their gut microbiome [39]. These studies have shown reductions of up to 5 log in the cecal counts of Campylobacter colonized chickens, including AMR Campylobacter strains [38]. As mentioned initially, the high variability reported in Campylobacter reduction might be dependent on a number of factors, such as the susceptibility of each strain to the applied phages [40][41][42][43], the route of phage administration [41,42], the dose and timing of application [38][39][40]42,44,45] or the development of phage-resistant Campylobacter mutants [40,41]. The use of polyphage therapy (campylophage cocktails) instead of single phages has been also studied for a broader host range [39,41,42,44]. Unfortunately, achieving complete Campylobacter elimination in broilers may be a difficult task. Nevertheless, the careful design and application of campylophage cocktails targeting different cell receptors (containing both group II and group III campylophages) has been suggested as the best approach to successfully combat Campylobacter, resulting in an efficient reduction in Campylobacter at the farm level, with a significant impact on food safety and public health [39,46].
Salmonella is the second zoonotic pathogen responsible for human gastrointestinal diseases. In fact, millions of human salmonellosis cases are reported worldwide every year, resulting in thousands of deaths. In the United States, Salmonella causes around 1.2 million cases every year, of which there are around 23,000 hospitalizations and 450 deaths [47]. In Europe, a total of 60,050 confirmed cases in humans were reported in 2021 by the European Surveillance System [28], reporting an increase of 14.3% in comparison with the previous year. Different serovars have been associated with salmonellosis, yet Salmonella Enteritidis, followed by Salmonella Typhimurium, has been the most common serovar related to FBO in humans worldwide [48], as well as in the EU [28]. 1 log reduction at day 35 of study [56] Although the use of antibiotics has been limited to therapeutics in Europe since 2012, the presence of resistant strains is being considered as a human and veterinary health concern. For instance, the data published by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (EDCD) in 2022 reported resistance of Salmonella against sulfonamides/sulfamethoxazole (30.1%), tetracyclines (31.2%), and ampicillin (29.8%) [57]. Moreover, resistance to ciprofloxacin was reported in 14.1% of the isolates, which was a slight increase compared with the previous report. Resistance to cefotaxime or ceftazidime was observed to be generally very low (<1%) among Salmonella spp. of the isolates. These antimicrobials represent the most important antimicrobial classes (fluoroquinolones and third-generation cephalosporins) used for the treatment of salmonellosis, and they have been classified by the WHO as the highest priority [58].
The current situation has encouraged the search for new alternatives, such as the use of phages against Salmonella [59]. Several studies showed phage biocontrol success in the poultry sector (Table 1), as it has been shown that phages reduced side effects compared to traditional antibiotic treatments due to their specificity [60]. At the field level, different publications have demonstrated the efficacy of phages in reducing Salmonella concentration in chickens. Zbikowska et al. infected chickens with Salmonella Gallinarum, and then animals were fed with a cocktail of phages, leading to a significant decrease in Salmonella in the organs as well as in the mortality of the chickens [61]. Similar results have been previously reported, showing that phages are a promising tool and an effective alternative to antibiotics [49,62]. Further, reductions of 1.53 log and 3.48 log of S. Enteritidis and S. Typhimurium, respectively, were reached after the application of a single dose of a phage cocktail [50]. Likewise, statistically significant differences in S. Enteritidis reduction after a later phage treatment demonstrated that the application of phages at late stages of broiler growth may be a promising measure for the control of this bacterium in future stages of the production chain [51].

Control of Listeria Monocytogenes in Animals
L. monocytogenes is a well-known pathogen responsible for listeriosis, one of the most serious food-and feed-borne zoonotic diseases worldwide. This pathogen can reach food products by contaminated raw materials or by cross-contamination during different steps of food processing [63,64]. Listeriosis in domestic animals is usually transmitted through the ingestion of contaminated feed and/or pet food, although it can also be transmitted through the upper respiratory tract mucosa, conjunctiva, and wounds [65]. Animal listeriosis is generally associated with encephalitis, abortion, septicemia, and mastitis in ruminants, but also in swine, horses, birds, rodents, fishes, and crustaceans, although an even wider range of animal species can also be affected.
Since animals act a reservoir and a main source of L. monocytogenes to humans, using the One Health concept, it would be reasonable to treat animals to control the introduction of Listeria into the food chain. However, the absence of harmonized regulations regarding the presence of L. monocytogenes at primary production has led to the low quantity of reported data [28]. Accordingly, as far as we know, there is still a lack of investigation into the use of phages for the control of L. monocytogenes in animals, and few examples have been found in the literature. The use of phage P100 has been proposed for the treatment of animals (including humans) infected with L. monocytogenes [66], but no further publications have been found regarding the application conditions or phage effectivity. Only one recent publication has demonstrated the potential therapeutic effect of phage LP8 against listeriosis in mice and the feasibility of a combined therapy to reduce the use of antibiotics in animals [67]. As more published research continues to focus on the application of Listeria phages in foods and food processing environments, this topic will be discussed in the sections below.

Control of Vibrio spp. in Aquaculture
Water also represents one of the most important methods of dissemination of AMR. Pathogenic microorganisms, such as Vibrio spp., occur naturally in water [68] and are the most important environmental human pathogen from aquatic and marine habitats [69,70]. In animals, vibriosis is responsible for important economic losses in turbot, salmonids, sea bass, and shrimps [71,72]. The incidence of all of these infections is rising, favored by the rising of sea water temperature due to climate change [68,73,74]. Additionally, as the global aquaculture production is increasing and is progressively growing into an intensive industry, the concentration of fishes in larger farms also may cause an increase in bacterial disease occurrence [71,73].
Although control and hygiene measures are important hurdles to the occurrence of an outbreak, antibiotics are still the most effective chemical agents for controlling Vibrio spp. Their abuse has caused the emergence of multidrug-resistant strains, and many Vibrios have already become highly resistant to most commercially available antibiotics [75][76][77]. With only a few antibiotics approved for aquaculture, this food source industry is continuously facing the threat of bacterial contamination. Furthermore, to mitigate antibiotic-resistant microorganisms, many countries have introduced strict antibiotic-handling programs, which include proper dosage of antibiotic treatment and objectives such as a 50% reduction in the use of antibiotics by 2030 in aquaculture [78]. Accordingly, the development of alternative biocontrol agents against Vibrio for aquatic hatcheries is also an urgent need, especially where vaccines cannot be applied. Some studies have shown the applicability of phages to reduce human pathogenic Vibrio spp. from aquaculture ( Table 2).
These studies have demonstrated the potential of phages controlling V. parahaemolyticus in vivo. For instance, the VP10 phage cocktail significantly reduced V. parahaemolyticus to undetectable numbers in mussels [79], and pVp-1 reduced bacterial growth by five orders of magnitude when phages were added into oysters' tanks [80]. Additionally, the VPG01 phage remarkably reduced the presence of V. parahaemolyticus in artificial seawater and in the aquatic crustacean Artemia franciscana [81].  Increased survival of larvae to >86% vs. 65% survival with antibiotics [87] 1 Water content (CFU/mL); 2 administered phage dose (PFU/mL); *AMR: antimicrobial-resistant strain.
For the last two decades, phages have been also studied for controlling animal vibriosis ( Table 2), most of them specifically targeting the fish pathogen Vibrio harveyi. It has been demonstrated that phages can reduce the mortality of infected shrimp larvae, from 75% (without phages) up to 20% [82]. More recently, a phage named Virtus has shown an important protective effect against mortality caused by V. harveyi on seabream larvae [83]. Phages have been also demonstrated to be useful weapons against V. anguillarum infections. The application of the phage CHOED increased Salmo salar survival rates in aquaculture conditions from 60% to 100% [84]. A similar result, namely a mortality rate less than 3%, was achieved using the VP-2 phage on zebrafish larvae as an infection model [85].
In aquaculture, phages may have additional advantages: Their specificity allows them to kill the target pathogenic Vibrio spp., while being unable to kill beneficial Vibrio spp. in fish microbiota. In addition, phages are especially easy to administer in water, and have the benefit of treating both the farm environment (water and facilities) and the farmed species [88]. This evidence suggest that phage therapy could be a viable alternative to protect and treat fish against these bacteria in different developmental stages, as well as preventing water-borne human Vibrio infections.

Phage Biocontrol at the Post-Harvest and Post-Slaughtering Stage
Pathogenic bacteria mostly contaminate the food products during the steps of harvesting, slaughtering, processing, and packing, and are becoming resistant to available antibiotics. Due to their potential, at present, there are many studies on post-harvest phage biocontrol interventions (direct food applications) for L. monocytogenes, Salmonella spp., C. jejuni, Vibrio spp., E. coli O157:H7, Cronobacter sakazakii, Shigella spp., and Staphylococcus aureus [89][90][91][92][93], among others. Below, we review some studies on the effectiveness of bacteriophage biocontrol of the selected foodborne pathogens on different food products.

Campylobacter
The consumption of contaminated raw and undercooked poultry meat is the major source of human campylobacteriosis [28]. The application of specific phages has been explored as a pre-harvest strategy to reduce Campylobacter colonization in broilers, as mentioned previously. Furthermore, although no commercial phage preparation is currently available for the biocontrol of Campylobacter in foods, some studies have also reported the efficacy of campylophages as a post-slaughter biocontrol strategy to reduce Campylobacter counts in different poultry products (Table 3) without affecting the remaining microbiota [33]. Three studies found a reduction of around one log in C. jejuni loads when artificially contaminated chicken skin samples were treated with phages and stored in refrigerated conditions (4-5 • C) [26,94,95]. The use of a high multiplicity of infection (MOI) of 10 3 has been suggested as the best approach to reduce Campylobacter load with no development of phage-resistant Campylobacter mutants [26].
The combination of phage treatment and freezing was shown to cause a further Campylobacter reduction of up to 2.5 log in chicken skin [26]. Under refrigerated tempera-tures, phage treatment was effective as a function of the campylophage receptor [95]: group II campylophages, which reversibly bind to host flagella, resulted to be unsuitable for Campylobacter biocontrol. Therefore, although the application of cocktails including both group II and III campylophages has been suggested to reduce Campylobacter colonization in broilers (Section 2.1), the use of only group III campylophage cocktails was proposed to successfully combat Campylobacter through post-harvest application [95]. While some authors reported negligible Campylobacter reduction in contaminated chicken meat [96], other researchers achieved a reduction of more than 1.5 log in raw and cooked beef [97] and chicken meat [98] after refrigerated storage for 1 and 2 days, respectively.
The ability of campylophages to reduce Campylobacter counts from chicken carcasses or food products may represent a promising approach to eliminating the risk of contamination from a finished product. Furthermore, the application of Campylobacter-specific phages could also provide an innovative alternative for surface sanitizing to reduce biofilms on food contact surfaces [33,99].    2.06 log reduction 2.51 log reduction [126] 1 Content in food (CFU/g or mL, unless specified); * MOI (multiplicity of infection: ratio between bacteriophage and bacterial load).

Salmonella
Many Salmonella species have in common the ability to form biofilms, which are being considered as a factor to explain the extreme persistence of Salmonella in food-processing environments. Consequently, although the food industry has evolved in recent decades, the risk of contamination during food processing remains high. Due to the implication of Salmonella on FBO, the interest in phage biocontrol has increased in the last year as a new method of microbiological control applicable to food pathogens. In this regard, phages have been postulated as an alternative that could be applied directly to food or during food production as disinfectants, due to their stability under abiotic conditions, null toxicity, and selectivity in antimicrobial activity [127].
Different approaches (Table 3) have been used to assess phage success in controlling Salmonella biofilms in foodstuff [128,129]. Phages have also been applied to food as a natural preservative to treat chicken carcasses against Salmonella that is non-recoverable after phage application, resulting in the elimination of the pathogen [59,94]. In the same way, Salmonella contamination from broiler and turkey carcasses rinses was reduced by 100% and 60%, respectively [100]. In addition, a reduction of 2.0 logs of S. Enteritidis in packaged chicken breast after treatment with a cocktail of phages was observed, and a reduction of 0.9 logs was reached in egg samples after phage treatment [101]. Another work assessed the effect of one phage against S. Enteritidis on different matrices, such as eggs and chicken meat. After 12 h of treatment, reductions of 1.79 log CFU/mL and 1.83 log CFU/mL were achieved, respectively [102]. In breast samples, a reduction of 2 log CFU/mL in the Salmonella contamination was observed after the application of 2 different bacteriophages [103]. In addition, several commercial phages against Salmonella for the poultry industry are available, showing promising results in Salmonella biocontrol [104][105][106][107]. In one of the studied cases, phage treatment was the most effective, in comparison with peracetic acid and cetylpyridinium chloride, in controlling Salmonella in chicken breast fillets under room temperature conditions [104].

Listeria Monocytogenes
Despite the low incidence of listeriosis, its high fatality rate makes it the most frequent cause of foodborne infection-related deaths [28,130]. The main route of human infection is the consumption of contaminated food and, specially, ready-to-eat (RTE) food products that do not require further cooking between production and consumption [28]. The extraordinary capabilities of L. monocytogenes to survive and grow in a wide range of temperatures, pH levels, acidic solutions, and salt concentrations [131][132][133], as well as its ability to form biofilms [134,135], make it very challenging to remove from processing facilities, equipment, and environments [136].
Phage biocontrol shows great potential to be used as a safety control approach at the postharvest stage of food production, in order to reduce the occurrence of L. monocytogenes in both the food-processing environment and the final food product (Table 3). Although few virulent Listeria-specific phages with potential for biocontrol have been characterized [137][138][139], some of them can infect not only the major L. monocytogenes serotypes, but also other species within the Listeria genus.
Several studies have assessed the effectiveness of commercial products (Phage Guard Listex™ P100 by Micreos B.V., and ListShield™ by Intralytix) and other Listeria-specific phages to control this pathogen in contaminated food products, with variable success. Treatment effectiveness is mainly influenced by the MOI ratio, i.e., the ratio between phage dose and Listeria load. High concentration of phages allowing treatments at high MOI ratios ensure successful contact between phages and their hosts, leading to a more efficient reduction in L. monocytogenes on RTE chicken breast roll [111], dry cured ham [114], raw salmon [108], soft cheeses [117], and lettuce [110]. More successful treatments were observed when phage application occurred during or directly after product contamination [118] and under refrigerated post-treatment storage conditions [112,114,120].
It has been observed that Listeria reduction was more effective in fruit juices, where phages can diffuse until they meet their host, than in fruit slices, where phages are immobilized and cannot contact their hosts through limited diffusion [119]. Similarly, more important reductions were obtained in melon products (slices and juice; pH 5.8 ± 0.1) than in pear products (pH 4.7 ± 0.2), suggesting that pH could be also a key factor contributing to phage effectiveness [119]. These results indicated that, as suggested by other studies, food-related factors, such as physical form, pH, food composition, and/or the presence of specific compounds or substances, may interact with receptors or cell surfaces and interfere with phage diffusion, receptor recognition, and/or binding [115,140].
The intrinsic properties (e.g., lytic spectra, stability, etc.) of the different Listeriaspecific phages directly affect treatment effectiveness. Better reduction was found on sliced apples after treatment with the cocktail Listshield [110] than with single phages [119], suggesting that the use of phage cocktails may contribute to better results [110]. Different reduction levels were also found after the application of different cocktails [118] and as a function of the target L. monocytogenes strain [115,141], underlying the importance of the lytic spectra of selected phages. Enhanced effectiveness of Listeria-specific phages has been reported when used in combination with other antimicrobials (e.g., bacteriocins or protective cultures) [108,109,112,113,116]. The application of phages as an innovative approach to eradicate L. monocytogenes biofilms in food processing environments and contact surfaces is another huge challenge that is currently being explored [120,140,142].
Overall, Listeria-specific bacteriophages and their cocktails could contribute, as an additional tool, to a multi-hurdle approach in order to safely reduce the occurrence and growth of L. monocytogenes in food products and food processing environments.

Human Pathogenic Vibrio spp.
Vibrio spp. are natural hosts in marine waters, and, consequently, are also naturally present in seafood. V. parahaemolyticus constitutes the major causative agent for seafoodborne gastroenteritis by the consumption of contaminated products [81,121]. On the other hand, although less frequent, V. vulnificus is also an opportunistic foodborne pathogen that may cause lethal septicemia [125]. As was previously mentioned, Vibrio infections are being controlled as emerging foodborne agents worldwide, and AMR is also increasing. Consequently, the need for alternative pathogen-control tools has become an urgent necessity. As in the case of Campylobacter, there are no commercial solutions for controlling Vibrio spp. yet. However, in recent years, research into this kind of solution has increased, according to the emergence of Vibrio FBO. There are many works focused on the development and application of phages, especially on V. parahaemolyticus control (Table 3).
For instance, the pVp-1 phage achieved a reduction of 6 log against a pandemic multidrug-resistant V. parahaemolyticus strain (CRS 09-17) when oysters were directly treated on their surfaces [80]. Other works have also reported an interesting effectivity when attempting to reduce V. parahaemolyticus counts in seafood products. Phage VPT02 showed about a 2 log drop in V. parahaemolyticus in raw fish flesh slices [121]. Similarly, the phages Vpp2 and OMN achieved reductions of about 2 logs in Manila clams and oysters, respectively [123,124]. Although more limited, the phages VPG01 and F23s1 have also demonstrated their capability, in solutions, to control the growth of V. parahaemolyticus in fresh fish and shrimps [81,122].
Regarding V. vulnificus, similarly, a phage cocktail has been also applied to reduce the load of V. vulnificus in eastern oysters from 10 6 to 10 1 CFU/mL [125]. A more recent study concluded that the VVP001 phage may be used to control V. vulnificus in a broad range of temperatures, ranging from −20 • C to 65 • C, showing a reduction of up to 2.51 logs of bacteria on abalone flesh [126].
These works have demonstrated that phages exhibit great potential as natural food preservatives for the biocontrol of potential Vibrio infections, as well as the prevention of contamination in diverse seafood-related circumstances, such as the storage and depuration steps of seafood [80] and the disinfection of seafood-processing equipment or utensils to prevent cross-contaminations [81].

Challenges of Using Phages for Food Safety
The use of phages as biocontrol tools has been gaining interest as a safety strategy in recent decades due to the emergence of AMR bacteria and the subsequent limited use of antibiotics in livestock and crops [143], thus remaining an interesting and natural alternative to combat bacteria. In terms of food safety, applications and advantages of phages have been already summarized in previous sections. However, although the results of the published studies appear to be promising, there are still some limitations that need to be addressed before their generalized use. To assist future phage-based real applications, pending issues and main challenges to be addressed shortly in future investigations are also reflected ( Table 4).
The high specificity of phages, their ability to overcome resistance, and their selfdosage can be both strengths and weaknesses. Phage specificity is a major issue for their effectivity as antimicrobials in biocontrol. Host tropism is mostly dependent on receptors based in the cell walls or bacterial capsules. In this situation, building a collection of phages or biobanks to confront most of pathogenic bacteria strains could be a huge and time-consuming undertaking and, depending on the species, direct hunting could be both faster and costless. Interestingly, biobanks could allow ready-to-use phages to be available that can recognize and lyse a battery of bacteria. However, this requires performing phagograms to quickly select the potential phages to be used. This process, known as "phage matching", could be easily performed with automated equipment, although is not common and the delay in determining the specific phage could be a problem. However, phage biocontrol can be effectively achieved as a customized treatment, which requires prior knowledge of the bacterial host and, most likely, phage hunting to select an efficient phage to control the target bacterium. Additionally, phages can be used as broad-range products by designing proper phage cocktails encompassing broad-range phages. Phage training (experimental evolution) or engineered phages could also help to broaden the host range and to obtain chimeric phages that could recognize multiple strains or species, although this may be detrimental to commensals. However, in food safety, and especially in the food industry, disinfectants to reduce bacterial burden are welcome, and phage-based products, including using phage-derived enzymes to eliminate bacterial biofilms, might be a promising solution as well. Indeed, phages encode several proteins with hydrolytic activity that can actively destroy the bacterial matrix composed of polysaccharide substances and can disrupt biofilms very effectively [144].  [155,156] Consumer acceptance • Unfounded fears and lack of contrastable information • Need for public awareness (provide education on the safety, efficacy, and ubiquity of bacteriophages to stakeholders (processors, consumers, etc.) [89] Another drawback of some phages is that they might be intrinsically unstable; therefore, some phage-based products might require some procedures to be followed to maintain their stability and, thus, their infectivity. Embedding phages within a material, such as nanoparticles, has been proposed to control phage release and targeted delivery, and could be useful for long-term storage and provision of commercial products that could be stable at different conditions [148]. In addition, other preservation methods, such as freeze-drying, could be another option for long-term storage of phages; they are much cheaper, making them an interesting solution for the industry. However, some phages are not able to maintain infectivity after processing, and encapsulation could be the preferred solution for food protection [149,150]. In this context, it is important to study the pharmacokinetics and pharmacodynamics of phages in the environment and in animals, in order to ensure their stability and potential immune responses. Additional in vivo assays are required to ensure the safety and efficacy of the phage biocontrol. In this view, phage administration routes and procedures should be deeply investigated to determine the outcome of the therapy [151].
Another point to be addressed is that phages can mobilize genetic material encoding resistant genes between strains, thus promoting the spread of AMR, including to nonpathogenic bacteria. Although phages are evolving entities in nature, and this transfer could certainly occur in their natural environment, for biocontrol purposes, lytic and perfectly characterized (sequenced) phages are always preferred to reduce potential gene transfer [152]. In addition, detailed analysis of each phage genome must be performed, as it provides useful information for the selection of the most suitable phages. In addition, understanding phage-host interactions will be of special interest to anticipate potential failure treatments, such as the emergence of phage-resistant bacteria. Interestingly, phages can overcome resistance, adapting to the new environment faster than their hosts. In addition, phage cocktails can be a solution to reduce the emergence of phage resistance [153].
Finally, to be used, any phage application must be in compliance with legislation. Nevertheless, the great variability of phage morphologies and diversity, their intrinsic evolving, and their self-replication nature in the presence of the bacterial host create a challenge for regulatory agencies due to their intrinsic evolvability [155], and highlight the problem of subjecting all phage-derived products to the same regulation and procedures. As seen, legislation on the use of phages is a complicated issue and will delay commercialization and routine use of this promising virus. However, regulatory agencies should provide rapid guidance on phage biocontrol to address the emergence of resistant bacteria, since an alternative to antibiotics is necessary [157].

Conclusions
Although it is clear that no therapeutic or preventive treatment can or should replace good hygiene practices in food production, progressively, more studies have demonstrated that phage application can be a leading approach to controlling important foodborne diseases. Considering their natural properties and advantages, phages can be used at all stages of the agriculture supply chain to control microbial pathogens. They can be employed in every step, from agriculture (primary production) to biosanitization of food processing facilities and biopreservation of foodstuffs. Moreover, the aforementioned challenges are expected to be answered as the issue of AMR becomes more pressing. The creation of a legal framework to allow different applications of phages in reality, including in food safety, is an especially pressing issue.
Author Contributions: All authors have substantially contributed to the writing and reviewing of this work. All authors have read and agreed to the published version of the manuscript.