Bacteriophages as an Alternative Method for Control of Zoonotic and Foodborne Pathogens

The global increase in multidrug-resistant infections caused by various pathogens has raised concerns in human and veterinary medicine. This has renewed interest in the development of alternative methods to antibiotics, including the use of bacteriophages for controlling bacterial infections. The aim of this review is to present potential uses of bacteriophages as an alternative to antibiotics in the control of bacterial infections caused by multidrug-resistant bacteria posing a risk to humans, with particular emphasis on foodborne and zoonotic pathogens. A varied therapeutic and immunomodulatory (activation or suppression) effect of bacteriophages on humoral and cellular immune response mechanisms has been demonstrated. The antibiotic resistance crisis caused by global antimicrobial resistance among bacteria creates a compelling need for alternative safe and selectively effective antibacterial agents. Bacteriophages have many properties indicating their potential suitability as therapeutic and/or prophylactic agents. In many cases, bacteriophages can also be used in food quality control against microorganisms such as Salmonella, Escherichia coli, Listeria, Campylobacter and others. Future research will provide potential alternative solutions using bacteriophages to treat infections caused by multidrug-resistant bacteria.


Introduction
Zoonotic pathogens cause problems all over the world, including diseases such as anthrax, brucellosis, bovine tuberculosis, hydatid disease, echinococcosis, trichinellosis, rabies, highly pathogenic avian influenza, Nipah/Hendra disease and bovine spongiform encephalopathy. In 2015, the WHO reported that more than 600 million people (1 in 10) worldwide fell ill as a result of foodborne infections [1,2]. According to the European Food Safety Authority (EFSA), the most frequently reported human foodborne diseases were campylobacteriosis and salmonellosis. However, the most dangerous pathogens for humans were identified as foodborne pathogenic bacteria found in livestock products, including enterohaemorrhagic Escherichia coli (EHEC; O157:H7), Shigella sp., Enterococcus spp. or Listeria spp. Multidrug-resistant pathogens isolated from human outbreaks, cattle, swine, and poultry were most often S. aureus, Streptococcus spp., Vibrio sp. and Yersinia spp. [3,4]. According to Niu et al. [5], these bacteria can also be transmitted to food products by direct contact with animals or indirectly by vectors such as insects, rodents, wild birds, or irrigation water.
The global increase in multidrug-resistant infections and antibiotic failures in control of pathogens has raised concerns in human and veterinary medicine. An official report of the European Food Safety Authority (EFSA) regarding zoonotic and indicator bacteria isolated from humans, animals, and food showed that a high proportion (28.6%) of human Salmonella strains were resistant to three or more antimicrobials, and 34.9% of E. coli strains isolated from pigs were resistant to more than six antibiotics [6].
There has been a marked increase in the antibiotic resistance of Gram-negative bacteria via a variety of mechanisms, such as antibiotic target modification, antibiotic degradation, and modulation of permeability through the bacterial membrane. These mechanisms have limited the development of novel antibiotics. The most resistant strains of bacteria are carbapenem-resistant Enterobacteriaceae, extensively drug-resistant (XDR) Pseudomonas aeruginosa, and XDR Acinetobacter baumannii. Understanding the mechanisms of resistance of multidrug-resistant bacteria is the main goal in the development of modern antibacterial agents [7].
Global livestock production is faced with an alarming increase in bacterial resistance, including among zoonotic pathogens. For example, Donkor et al., [8] showed higher antimicrobial resistance in livestock than in humans, with animal E. coli isolates exhibiting high levels of resistance to tetracycline and penicillin. This has led to renewed interest among scientists to develop alternative methods to antibiotics, including the use of bacteriophages, since the beginning of the 21st century [9].
Widespread multidrug resistance among bacteria necessitates the search for alternative methods of controlling infections, including pre-and probiotics, vaccines, bacteriophages, nanoparticles, antimicrobial peptides (AMPs) and others. An example is the use of bacteriophages to reduce or eliminate pathogenic bacteria in livestock production, as biocontrol agents to control foodborne pathogens and to reduce contamination on food-contact surfaces [9]. An important contribution to research on the use of bacteriophages to control bacteria, including zoonotic pathogens, is the development and implementation of new legal regulations in the EU regarding restrictions or complete bans on the use of selected groups of chemotherapeutics in individual sectors of animal production. An example of such legislative action is the EU Council Directive 2019/6 [10] coming into force in January 2022.

General Characteristics of Bacteriophages
Due to the widespread nature of bacteriophages (phages) associated with crops, live animals, and human intestinal environments, humans have direct and indirect contact with them. Many studies have demonstrated the common presence of bacteriophages in various fermented foods, such as yogurt and cheese. The application of specific bacteriophages to foods helps to reduce foodborne pathogenic bacteria [5].
Bacteriophages are bacterial viruses, causing complete lysis of a susceptible bacterial culture [11]. Interactions between phages and bacteria can be regarded as parasitism, as most virulent phage replication necessarily results in bacterial death. Certain interactions can be termed mutualistic, while some temperate phages encode benefits for the phenotypic properties of the host bacteria [12] According to Batinovic et al. [13], the prevalence of bacteriophages in the environment has been a natural phenomenon for billions of years, resulting in a balance of commensal and pathogenic bacteria. Phages and bacteria are the oldest and most ubiquitous microorganisms on Earth, likely having originated approximately 3 billion years ago [14,15].
Phages are prevalent in a variety of environments, including water, forest groundcover, food products, wastewater, and animal and human waste [16]. Bacteriophages have also been detected in commercial products, such as sera and human vaccines, as well as inside the human mouth (dental plaque and saliva) and in the gastrointestinal tracts of animals and humans [17].
Although bacteriophages may be present autonomously outside the host, all phages require the bacterial cell as a host for multiplication. Most phages are highly specific for host cell surface receptors such as receptor binding proteins (RBPs) or LPS [18,19].

History of Bacteriophages
Bacteriophages were first discovered more than 100 years ago by two microbiologists, Frederick Twort from England and the French Canadian Felix d'Herelle [20,21]. The first experimental and successful phage therapy was carried out by D'Herelle in the control of fowl typhoid in chickens (95-100% survival) [22]. He also coined the term 'bacteriophage', meaning 'bacteria eater'. Finally, in 1940, electron microscopes were used to identify the viral nature and morphology of phages [23]. Bacteriophages have been used in various types of therapies in humans, e.g., in dermatological, ophthalmological, urological, paediatric, otolaryngology and surgical infections. The significant therapeutic success of these treatments had a major impact on the development of phage therapy in the pre-antibiotic era. This was crucial, as the only treatment available in the first two decades of the 20th century was serum therapy (e.g., for pneumococci or the diphtheria bacterium), so bacteriophage therapy began to dominate in human medicine [24].
The discovery of the antimicrobial properties of Penicillium notatum in 1928 by Alexander Fleming culminated in the successful development of the first major antibiotic, penicillin, in 1941 [25], which marked the beginning of the antibiotic era and naturally inhibited the development of bacteriophage therapy.
At present, as bacterial resistance to antibiotics is increasing significantly worldwide, phages are one of the factors with potential to replace them [26]. The best known bacteriophage centres in the world are the Eliava Institute of Bacteriophages, Microbiology, and Virology (EIBMV) of the Georgian Academy of Sciences, in Tbilisi, Georgia, and the Hirszfeld Institute of Immunology and Experimental Therapy (HIIET) of the Polish Academy of Sciences, in Wroclaw, Poland. Both institutes offer phage therapy against many bacterial and fungal pathogens, e.g., Staphylococcus spp., Klebsiella sp., Proteus sp., E. coli, and Pseudomonas sp., as well as other enteric pathogens [27][28][29].

Classification of Bacteriophages
Bacteriophages are the most widespread life forms on Earth. By 2018 year more than 650 strains of bacteriophages had been deposited in the American Type Culture Collection (ATCC) and >27,000 bacteriophage nucleotide sequences had been deposited in the International Nucleotide Sequence Database Collaboration (INSDC) [30]. The total number of these bacterial viruses has been estimated at 10 32 , which is 10 times the number of characterized bacteria. In water, the total count of bacteriophages has been estimated at 10 4 to 10 8 virions/mL −1 [31].
The classification of bacteriophages is based on the type of nucleic acid (ssRNA, dsRNA, ssDNA, dsDNA), the structure of the capsid (e.g., helical, pleomorphic, icosahedral, filamentous/thread-like, complex/polyhedral), which is built of structural proteins, and their life cycle, bacterial target, and site ( Figure 1). The phage taxonomy criteria applied by the International Committee on Taxonomy of Viruses (ICTV) were nucleic acid composition and virion morphology [9]. In 2015 the Bacterial and Archaeal Viruses Subcommittee (BAVS) classified phages into 873 species, 204 genera and 14 subfamilies [32]. The classification of bacteriophages is still ongoing, and in 2018 the ICTV presented a new classification of these bacterial viruses into 142 families, 81 subfamilies and 4978 species [9]. Most bacteriophages (96%) belong to the order Caudovirales, which is grouped into three main families: Myoviridae, Podoviridae and Siphoviridae [32]. Most bacteriophages contain double-stranded DNA, and the nucleic acid is coated with a protein capsid. Some phages have an additional layer (envelope) [17]. As new bacteriophages are continually being detected, their classification is constantly modified. The latest classification of viruses, based on the virus taxonomy proposed by the ICTV, was presented in our previous paper [33].

Life Cycles of Phages
The life cycle of phages is an important element of infections of bacteria. Phages can be categorized into types based on their virulence: lytic (virulent, productive) and lysogenic (temperate, dormant). Virulent phages follow a lytic cycle in the bacterial cell and lyse it to release a newly created population of phages [34].
The lytic cycle includes the adsorption, penetration, biosynthesis, assembly and release of bacteriophages from the infected bacterium. During this process many phages use specific proteins located on the surface of the bacterial cell as receptors. During the adsorption phase, the bacteriophage adheres to the bacterial cell, and phage proteins bind to specific receptors, such as teichoic and lipoteichoic acid for Gram-positive bacteria or LPS for Gram-negative bacteria [35]. The next phase, penetration, consists of destruction of the bacterial wall by bacteriophage enzymes and insertion of the genetic material into the bacterial cell. This is followed by the formation of capsid structures for nucleic acid and protein replication, accompanied by inhibition of replication of bacterial DNA. The phage genetic material is transcribed in the bacterial cell by RNA polymerases to produce mRNA, which supresses host intracellular synthesis as a consequence of bacteriophage multiplication [36]. Tens, hundreds or thousands of replicated phages are released by means of lysis of the bacterial cytoplasmic membrane by a phage protein (holin) and the formation of pores by endolysin encoded by double-stranded phage DNA and peptidoglycan hydrolases. The duration of the entire lytic cycle may be 20-40 min or up to 1-2 h [9,37].
Lysogenic infection via phages involves integration of their genetic material into the chromosome of the infected bacteria (prophage), which does not destroy the bacterial cell or produce a new population of bacteriophages. It leads to the integration of the phage genetic material with the bacterial DNA and its transmission into a new population of bacteria. This kind of bacteriophage is called a temperate phage, and in cells carrying a prophage it is referred to as lysogenic. Nevertheless, the viral prophage, also called an endogenous phage (a latent form of phage), can become activated by abnormal environmental conditions and other external stress factors that can damage the bacterial genetic material, including sunlight, UV radiation, some alkylating cytostatics (chlorambucil, cyclophosphamide, ifosfamide, estramustine or chlormethine) or mutagenic antibiotics such as mitomycin C. In some cases, the prophage is excised incorrectly from the chromosome, taking with it neighbouring bacterial genes. This is one of the main means of horizontal gene transfer (HGT) among bacteria, which is also one of the main methods in molecular biology [9]. Phages which have been defined as temporary include E. coli Lambda [38], with activity against E. coli and other Enterobacteriaceae; phage Mu, specific for Salmonella, Citrobacter and Erwinia; MM1 Streptococcus pneumoniae; and φ11 S. aureus [39].
In another type of life cycle involving chronic infection, bacteriophages infect the bacterial cell, in which new phage populations arise without destroying the bacteria. The chronic infection lifestyle is found in rod-shaped (filamentous) single-stranded DNA phages and in plasmaviruses that infect mycoplasmas. In the chronic infection lifestyle,

Life Cycles of Phages
The life cycle of phages is an important element of infections of bacteria. Phages can be categorized into types based on their virulence: lytic (virulent, productive) and lysogenic (temperate, dormant). Virulent phages follow a lytic cycle in the bacterial cell and lyse it to release a newly created population of phages [34].
The lytic cycle includes the adsorption, penetration, biosynthesis, assembly and release of bacteriophages from the infected bacterium. During this process many phages use specific proteins located on the surface of the bacterial cell as receptors. During the adsorption phase, the bacteriophage adheres to the bacterial cell, and phage proteins bind to specific receptors, such as teichoic and lipoteichoic acid for Gram-positive bacteria or LPS for Gram-negative bacteria [35]. The next phase, penetration, consists of destruction of the bacterial wall by bacteriophage enzymes and insertion of the genetic material into the bacterial cell. This is followed by the formation of capsid structures for nucleic acid and protein replication, accompanied by inhibition of replication of bacterial DNA. The phage genetic material is transcribed in the bacterial cell by RNA polymerases to produce mRNA, which supresses host intracellular synthesis as a consequence of bacteriophage multiplication [36]. Tens, hundreds or thousands of replicated phages are released by means of lysis of the bacterial cytoplasmic membrane by a phage protein (holin) and the formation of pores by endolysin encoded by double-stranded phage DNA and peptidoglycan hydrolases. The duration of the entire lytic cycle may be 20-40 min or up to 1-2 h [9,37].
Lysogenic infection via phages involves integration of their genetic material into the chromosome of the infected bacteria (prophage), which does not destroy the bacterial cell or produce a new population of bacteriophages. It leads to the integration of the phage genetic material with the bacterial DNA and its transmission into a new population of bacteria. This kind of bacteriophage is called a temperate phage, and in cells carrying a prophage it is referred to as lysogenic. Nevertheless, the viral prophage, also called an endogenous phage (a latent form of phage), can become activated by abnormal environmental conditions and other external stress factors that can damage the bacterial genetic material, including sunlight, UV radiation, some alkylating cytostatics (chlorambucil, cyclophosphamide, ifosfamide, estramustine or chlormethine) or mutagenic antibiotics such as mitomycin C. In some cases, the prophage is excised incorrectly from the chromosome, taking with it neighbouring bacterial genes. This is one of the main means of horizontal gene transfer (HGT) among bacteria, which is also one of the main methods in molecular biology [9]. Phages which have been defined as temporary include E. coli Lambda [38], with activity against E. coli and other Enterobacteriaceae; phage Mu, specific for Salmonella, Citrobacter and Erwinia; MM1 Streptococcus pneumoniae; and ϕ11 S. aureus [39].
In another type of life cycle involving chronic infection, bacteriophages infect the bacterial cell, in which new phage populations arise without destroying the bacteria. The chronic infection lifestyle is found in rod-shaped (filamentous) single-stranded DNA phages and in plasmaviruses that infect mycoplasmas. In the chronic infection lifestyle, phages are gradually eliminated from the bacteria over a long period without destruction of the cell [40].

The Spectrum of Use of Bacteriophages
The specificity of phage activity means that they infect only the bacteria specific for them (called the host) via external receptors, which determines the phage host range. Therefore, the use of phage therapy relies on a detailed and accurate characterization of the bacteria, including pathotypes and serotypes. Bacteriophages can be used in a variety of forms and methods to control and eliminate bacteria, including therapy, food protection and sanitation procedures [1,9]. Examples of the scope of the use of bacteriophages are presented in Table 1. Mainly to Vibrio spp., less to Edwardsiella spp., Lactococcus spp., Pseudomonas spp., Aeromonas spp., Flavobacterium spp. [49] Bacteriophages can potentially be used as biological control agents, especially in the reduction and elimination of bacterial contamination in foods, e.g., by Salmonella, Listeria monocytogenes, Campylobacter spp. or E. coli O157:H7 [15,50]. The high efficacy and safety of bacteriophage therapy is due in part to their specificity for selected bacteria: a single species, serotype, or strain. This is beneficial because the commensal gut microbiota is not destroyed. Another advantage is that, due to the self-replication of bacteriophages at the site of application, repetition of the application is often unnecessary. In many cases, no side effects of clinical treatment are observed, indicating a high level of safety that has been confirmed in many studies [51]. However, the application of bacteriophages in live animals or humans induces a cellular immune response, which could lead to the inactivation of phages, rendering them ineffective in eliminating bacteria [52][53][54].

Bacteriophage Interactions during Phage Therapy
Bacteriophages are regarded as the most applicable ecological and alternative means of elimination of pathogens (control and prevention of infections) due to their natural origin and numerous advantages, including the following: • lysis of bacteria usually highly resistant to antibiotic therapy, living in a biofilm; • high degree of safety for commensal and symbiotic flora; • possibility of use with other bacteriophages as a cocktail or with other antibacterials; • complete biodegradability of bacteriophages, making them safe for the organism and the environment [17].
However, phage therapy may carry a risk of immunological reactions, which is linked to the protein structures of bacteriophages. The immune response to bacteriophages depends on the location of the bacterial infection and the route of administration of the phages. The activity of bacteriophages also relies on their ability to penetrate epithelial cells and potentially spread to the bloodstream, lymph and internal organs such as the lung, liver, kidney and brain [61]. Bacteriophages can activate dendritic cells to synthesize pro-inflammatory factors (including IL-6, IL-1α, IL-1β and TNF-α) and to induce changes in the expression profile of these cell surface proteins and activation of the NF-κB signalling pathway [62].
The results of many studies confirm that bacteriophages can be phagocytosed by mammalian cells [63]. For example, Geier et al. [64] demonstrated rapid removal of wild-type phage λ from the circulatory system in humans. According to the authors, phagocytosis via immune cells is the main process of elimination of bacteriophages in mammals, and this mechanism takes place during lysis of bacteria by bacteriophages, which increases the activity of phagocytic cells, including PMN cells. The higher number of neutrophils at the site of infection is necessary to remove phage-resistant bacteria; this neutrophil-phage cooperation process has been confirmed in the resolution of P. aeruginosa infections [53,65]. However, some studies [66] have confirmed that bacteriophages can also express anti-inflammatory properties by decreasing the expression of TNFα and monocyte chemoattractant protein-1, which reduces ROS production by neutrophils and protects the epithelia against damage.
Some bacteriophages can also be a natural component of the intestinal microbiota and consumed food [67]. The oral administration of phages against Staphylococcus, Klebsiella, Escherichia, Proteus and Pseudomonas also induces the production of antibodies [68]. There has been no evidence of immunological disorders following phage ingestion per os at any concentration [69]. Topical application of phages to animals and humans also caused no side effects [70].
Minor problems have been observed in the case of other internal organs and blood vessels, which are not natural environments for phages. Here the immunogenic and immunomodulatory effects of phages can be observed. Bacteriophages can have non-specific effects on the immunological functions of various immune cells, including PMNCs, as well as on cytokine production and the induction of specific antibodies against non-phage antigens [71]. For example, resident liver macrophages (also called Kupffer cells) are able to eliminate bacteriophages by phagocytosis four times faster than spleen macrophages. The natural innate immune response is usually sufficient to eliminate pathogens before the activation of adaptive immune mechanisms. Bacteriophages can activate immune mechanisms and thus affect the metabolic activity of immune cells. However, bacteriophages can inhibit the production and release of reactive oxygen species in response to pathogens, which could decrease innate antibacterial immunity. [72]. Phages can induce antibodies that neutralize them, which can inhibit the antibacterial effect of phages in the form of lysis of targeted bacteria [71,73]. It is not currently clear how long this type of antibody will remain in the body, as knowledge of the kinetic aspect of bacteriophage activity is insufficient. Moreover, the titre of these antibodies depends on many factors, including the route of application (local application causes a minor increase in antibodies) and its frequency [74]. Some information about the influence of bacteriophages on immune responses in animal's model has been presented in Table 2.  [82] Antiphage antibodies are probably one of the most important factors influencing the efficacy of phage therapy. However, the activation of the production of neutralizing antibodies by phages need not be a problem during the initial phase of treatment of bacterial infections, because bacteriophage activity is much faster than the production of phage-neutralizing antibodies [27]. However, these antibodies can affect the efficacy of treatment during the second phase of therapy. This necessitates the implementation of additional solutions, such as the following: • repeating phage administration two or more times, because bacteriophages can multiply at the site of application during infection of the host bacteria; • increasing the phage concentration in the solution, because a high level of phages protects against complete destruction by neutralizing antibodies; • using different phages, especially during the second and subsequent cycles of application during treatment, because resistance differs from one phage to another [27].
In addition to the increase in neutralizing antibodies during phage therapies, the concentration of class M and G immunoglobulins increases as well and continues to increase with subsequent applications of phage preparations [83,84].
Besides activating humoral response mechanisms, phages also play a significant role in the modulation of cellular immunity against them. For example, s.c. application of MS-2 phages induced a hypersensitivity reaction in guinea pigs [85]. It has been suggested that the cellular response plays only a minor role in phage inactivation, as observed in the case of phage T7 in T cell-deficient mice [79]. In another study [86], phages had an immunosuppressive effect by suppressing the activation of T lymphocytes during the development of transplantation tolerance.
While bacteriophage therapies have been an effective tool in control of bacterial infections in various animal species, phages are also currently used for typing and diagnosis of specific bacterial species and for control of foodborne pathogens in food.

Commercial Products with Bacteriophages for Elimination of Foodborne Zoonotic Pathogens
Foodborne infections are the most important global health problem, contributing significantly to hospitalizations and deaths worldwide despite many advances in pathogen surveillance. Traditional food sanitation techniques using antimicrobial methods (including pasteurization, high pressure, irradiation or chemical treatment) can reduce pathogens in foods in varying degrees. However, these methods may damage equipment and adversely affect the organoleptic qualities (and possibly the nutritional value) of foods. The most important problem with using chemicals is that they eliminate 'good' microbes, which are beneficial in natural preservation of foods [87]. Therefore, it seems preferable to use an effective natural and ecological alternative such as bacteriophages for biocontrol of foodborne pathogens. Bacteriophages are mainly used in three sectors of the food industry to ensure food safety: primary production, biopreservation and biosanitization. As components of commercial products, they are currently finding application in the elimination of pathogens from food products of animal origin (meat products, milk and dairy products) or plant origin (fresh fruits and vegetables).
The number of commercial bacteriophage products approved for use in food safety in various countries is continually increasing. Many commercial companies around the world have shown interest in information about the use of bacteriophages as antibacterial tools to control foodborne pathogens, e.g., in the United States (AmpliPhi Biosciences (VI, USA), Enbiotix (Boston, MA, USA); Intralytix), the United Kingdom (Novolytics, Sarum Biosciences and Fixed Phage, Bolton, UK), EU countries such as France (Pherecydes Pharma, Romainville, Ile-de-France, France) and Portugal (Technophage and InnoPhage, Lisbon, Portugal), and other countries [88]. Detailed information about commercial bacteriophage products used for biocontrol of foodborne pathogens in food is presented in Table 3.

Advantages and Disadvantages of Bacteriophage Therapy
Phages have several advantages over antibiotics as therapeutic agents, such as activity against all types of bacteria, including MDR-pathogens. Their narrow antibacterial spectrum (which protects the natural microbiome), the low level of side effects, and their extensive distribution when administered systemically are also worth noting. They also may exert an effect on the inflammatory response, and their low production cost and high efficacy are significant benefits [31,99]. Many studies have confirmed the beneficial effects of the use of bacteriophages, shown as follows: Bacteriophages show high specificity for their target pathogens and kill only pathogens without destroying the physiological saprophytic flora; the narrow host range of phages is also a useful feature in prophylaxis of infections caused by enteric bacteria [100].
The distribution of phages in the body following systemic administration is much more extensive than in the case of antibiotics, in part due to the lack of or very low level of resistance of bacteria [31].

•
Because the mechanism of action of phages against the host bacteria is different to that of antibiotics, they are highly effective against many pathogens, especially against multidrug-resistant bacteria [36]. • Phages replicate at the site of infection even after a single application, because they multiply inside the bacterial cell and therefore are released at the site of infection [101].

•
Bacteriophages are resistant to stress factors during food production [91]. • Phage therapy is theoretically cheaper than antibiotic therapy due to the simplicity of production [99]. The unit costs of production as well as the costs of isolation and characterization are comparable or even lower than the costs of classical chemotherapeutic products [102].

•
There is no withdrawal period in livestock due to the lack of residue in tissues as soon as therapy is completed [103].

•
There are no side effects or allergic reactions because most bacteriophages consist mainly of proteins and genetic material (DNA or RNA).
However, in addition to the positive effects of phage therapies, widespread use of bacteriophages is limited by obstacles such as the following:

•
Due to their high specificity for a single type of bacteria, bacteriophages have a narrow host range [104]. • Bacteriophages may neutralize antibodies, which may prevent a portion of the administered phage dose from adhering to the target bacteria [104].

•
Only lytic phages are admissible in phage therapy because lysogenic (temporary) phages may be a source of horizontal transfer of bacterial toxins or antibiotic resistance [31].

•
The duration of survival of phages is varied, depending in part on the presence of the host bacteria. Their activity is also influenced by the environment within the organism in which it is administered, and therefore the survival of phages must be monitored at the site of administration in order to assess their antimicrobial activity [99]. • Information about the kinetics of phages remains insufficient, especially the degree of adsorption, the number of replications necessary for a therapeutic effect, the latent period, and their elimination from the body by phagocytic cells [106].

Bacteriophage Efficacy in Experimental Models
There are many methods of application of phages in therapies for humans and animals, including intraperitoneal, subcutaneous or intramuscular injection or oral, intragastric, rectal, topical or intranasal administration. Forms of administration of phages during therapy include sprays, aerosols, lozenges, compresses, mouthwash, suppositories, throat rinses, bandages, eye or ear drops and tampons [107]. In many studies in humans and animals, the form of application and type of administration has been associated with the type and location of the disease. In earlier studies [108][109][110][111], the best therapeutic effect was observed after direct application of phages to the target bacteria, as in the case of bacterial dysentery caused by Shigella; intestinal dysbacteriosis caused by E. coli and Proteus spp.; lung and pleural infections caused by Staphylococcus; suppurative skin infections caused by Pseudomonas, Staphylococcus, Klebsiella, Proteus, and E. coli; and infections of the skin or nasal mucosa caused by Klebsiella spp.
Some studies have explored the use of phages for control and treatment of neonatal enterotoxigenic E. coli infections in cattle, poultry and pigs [40,112]. Bacteriophages have also been used in controlling systemic infections with foodborne pathogens, including Salmonella spp., E. coli, Campylobacter spp., Vibro spp., Pseudomonas aeruginosa, and other pathogens, such as Staphylococcus spp., Streptococcus spp., Klebsiella spp., Acinetobacter spp., and even Mycobacterium spp. These experiments were carried out in experimental mouse or rat models, as well as in chickens, rabbits, calves, pigs and sheep. Examples of the effects of experimental phage therapies in different animal species and in the control of various pathogens are presented in Table 4.

Conclusions
To sum up, bacteriophages have many properties indicating their potential suitability as therapeutic or/and prophylactic agents. Future research on the scope of phages will provide a good picture of their potential to treat infections caused by multidrug-resistant bacteria. However, as bacteriophages are essentially 'living' drugs, the study of their use for therapy or biocontrol spans from purely clinical observations to molecular analysis to considerations of immunology and ecology. Due to the antibiotic resistance crisis, there is a compelling need for alternative safe and selectively effective antibacterial agents.

Conflicts of Interest:
The authors declare no conflict of interest.