Bacteriophages are a group of viruses widely distributed in nature whose life cycle is strictly associated with the bacterial cell. They are known as bacterial parasites because they lack the cell structure and enzyme systems necessary for food uptake, protein synthesis or construction of new particles, and as incomplete organisms can only replicate in a live cell.

Bacteriophages were discovered by Twort (1915) as unidentified molecules that inhibit bacterial growth, but in 1917 D’Herelle was the first to isolate and characterize phages, and he also developed the first phage therapy against fowl typhoid induced by Salmonella Gallinarum in chickens [1]. Positive results of the use of bacteriophages in fighting bacterial infections have contributed to the development of research on the potential use of viruses that destroy bacteria in treatment of diseases in both human and animals [2, 3].

Taxonomy of bacteriophages and life cycles

The criterion of taxonomy of bacteriophages applied by the ICTV (International Committee on Taxonomy of Viruses, EC 48, Budapest, Hungary, August 2016) is based mainly on genome type and virion morphology. The ICTV report, based on genomic and proteomic-based methods, was used by the BAVS to classify phages into 873 species, 204 genera and 14 subfamilies in the 2015 taxonomy release [4,5,6]. The basic classification of the viruses is shown in Table 1. It should be emphasized that the vast majority (about 96%) of known phages belong to the Myoviridae, Podoviridae and Siphoviridae [7, 8].

Table 1 The basic classification of the viruses based on Virus Taxonomy: 2016 Release EC 48, Budapest, Hungary, August 2016

Their fundamental characteristic is the presence of one type of nucleic acid as a carrier of genetic information and a capsid built from structural proteins. In terms of DNA structure, phages can be divided into three groups: those containing DNA in the form of a double helix, those with a single strand of DNA, and phages containing RNA. Most known bacteriophages have a genome consisting of double-stranded DNA. Two types of bacteriophages are distinguished on the basis of capsid symmetry: isometric (polyhedral) and helical (spiral).

Estimates suggest that bacteriophages are the most abundant life forms on Earth. By 2017 more than 25,000 bacteriophage nucleotide sequences had been deposited in INSDC databases [5, 9]. The common occurrence of bacteriophages is a significant factor facilitating their acquisition and characterization of their suitability for combating bacterial infections. Phages are isolated from all natural environments, including wastewater, human and animal waste, natural water bodies, soil, forest groundcover, food products, and other microorganisms [10,11,12].

Replication of bacteriophages is similar in many ways to that of eukaryotic viruses. Both involve adsorption, penetration, replication of nucleic acids, formation of virions, and their release from the host cell. Bacteriophages are specifically associated with a particular bacterial strain and exhibit strong bactericidal activity against Gram-positive and Gram-negative bacteria. Some phages display specific affinity for single types of bacteria, while others have a broad range of activity. Their specificity and range of activity is determined by the presence of receptors located on the surface of bacterial cells, among which we can distinguish LPS fragments, fimbriae and other surface proteins [8, 13,14,15].

We distinguish two types of activity against the bacterial cell: lytic activity, which is characteristic of virulent phages, and lysogenic activity, involving integration of the genetic material of the bacteriophage with the bacterial chromosome and replication as part of the bacterial DNA, resulting in the appearance of a prophage [15].

The lytic cycle of bacteriophages consists of adsorption, which involves adhesion to the bacterial cell, and binding of phage proteins to previously recognized receptors on the bacterial cell surface, such as teichoic and lipoteichoic acid for Gram-positive or LPS for Gram-negative bacteria [14]. The penetration phase involves rupture of the cell wall by the bacteriophage enzymes and penetration of the genetic material into the host cell. Next is the eclipse phase, involving replication of nucleic acid and proteins constituting the structural part of the capsid, while replication of the bacterial DNA is inhibited. This is followed by the formation and maturation of the bacteriophage, lysis of the bacterial cell and the release of daughter phages capable of infecting other cells [8] (Fig. 1). Examples of bacteriophages undergoing the lytic cycle are phages T1 and T4 [16].

Fig. 1
figure 1

Different types of bacteriophage infection [8]

The lysogenic cycle involves direct integration of genetic material with the bacterial chromosome, integration with the host genome and formation of the prophage. The replication of the bacteriophage is blocked and its genome enters a state of latency. This state can be interrupted spontaneously or as a result of activation by sunlight, UV radiation, alkylating agents, or certain antibiotics, such as mitomycin C [8, 11] (Fig. 1). Examples of bacteriophages with a lysogenic cycle include λ Escherichia coli; Mu, with activity against E. coli, Salmonella, Citrobacter and Erwinia; MM1 S. pneumoniae; and φ11 S. aureus [12, 16].

Depending on environmental conditions and the type of bacterial cell, there are several different pathways of bacteriophage infection, including chronic infection, pseudolysogeny and abortive infection (Fig. 1). Not all of these cycles end with the death of the bacterial cell and replication of phage particles. In many cases daughter virions are produced without induction of lysis of the bacterial cells, and thus viral particles are not released outside the cell [8, 17, 18].

Bacteriophages for control pathogens

The most common bacteria inducing foodborne infections in humans include bacteria of the genera Salmonella and Campylobacter and E. coli. According to the 2015 EFSA report on resistance to antibacterial agents in selected zoonotic bacteria (Salmonella and Campylobacter), indicator bacteria (E. coli and Enterococcus spp.), and other bacteria isolated from poultry and from food, a considerable percentage of the isolates posing a threat to humans and animals are resistant to available antibiotics, partially as a result of their widespread use in treatment of disease in humans and animals. The use of bacteriophages to eliminate pathogens seems quite promising, especially as they are present in every ecosystem and number 1031, which is more than 10 times the number of characterized bacteria [11, 19, 20].

The effectiveness and safety of phage therapy in comparison to antibiotics is partially due to the specificity of bacteriophages for particular bacteria, manifested as the ability to infect only one species, serotype or strain. This mechanism of action does not cause destruction of the commensal intestinal flora. Self-replication of bacteriophages takes place during treatment, which eliminates the need to apply them repeatedly. Another advantage of phages is that they cannot bind to and replicate in eukaryotic cells, which causes a decrease in their titre, correlated with a marked reduction in the number of pathogenic bacteria inducing a given infection in the organism. An equally important advantage is that phages are not toxic, because most of them are composed mainly of proteins and nucleic acids [21].

Despite its numerous advantages, the use of phage therapy is substantially limited, partly because single bacteriophages cannot be used to combat broad-spectrum infections. In many cases complex identification and characterization of the aetiological agent is necessary. Moreover, not all bacterial viruses meet the criteria for use in treatment, particularly lysogenic phages, which encode genes of bacterial toxins and thereby transform harmless bacteria into pathogenic ones. They can also be involved in transferring drug-resistance genes among bacteria. Another adverse phenomenon in phage therapy is that phages can be cleared by the reticuloendothelial system, reducing their half-life in the organism and limiting the effectiveness of treatment [18, 19, 22].

The increased use of treatment with bacteriophages is determined by their ability to lyse infected bacteria and mutate resistant bacteria, as well as by the high specificity of phages for particular bacteria. A vast number of infections in humans are induced by multi-drug resistant hospital strains of bacteria and by bacteria which have acquired resistance traits in the natural environment. Phage therapy has found application in treating bacterial infections in dermatology, stomatology, otolaryngology, ophthalmology, gynaecology, paediatrics, gastroenterology, urology and pulmonology [23]. The use of bacteriophages in treating infections in humans has had a high success rate (about 85%), particularly in the case of mixed infections induced mainly by Staphylococcus aureus, Klebsiella, Escherichia coli, Proteus, Pseudomonas, Enterobacter and vancomycin-resistant Enterococci [24, 25].

Application of phages in biocontrol and therapeutic design

Phage therapies are also an effective tool in eliminating bacterial infections in various species of animals. Bacteriophages have also proven successful in treating diseases in poultry. One of the objectives of phage therapy in animals is to assess the suitability of bacterial viruses for control of pathogens having an important influence on animal productivity and health. Phages used in treatment have been effective in preventing infections and in treatment of colibacteriosis in poultry [26]. Positive results, with a high success rate in eliminating pathogens, have also been obtained in combating infections induced by various Salmonella serotypes in gamefowl, such as Enteritidis and Typhimurium [27,28,29,30,31,32], as well as campylobacteriosis in poultry, particularly infections induced by Campylobacter jejuni and C. coli [33]. The effectiveness of phage therapy has also been confirmed in infections of broiler chickens by anaerobic Clostridium perfringens during the course of necrotic enteritis [34].


The therapeutic effectiveness of phages is determined by their high lytic titre, the form and type of application, and the application period. Long-term use of phages in poultry has proved to be moderately effective in reducing the number of Salmonella pathogens colonizing the digestive tract [27]. However as shown by Fiorentin et al. [28], single oral application of a cocktail of phages (CNPSA1, CNPSA3 and CNPSA4) at a dosage of 1011 PFU decreased the occurrence of Salmonella Enteritidis strains by 3.5 log units. The authors confirmed that applying a single dose of a bacteriophage suspension with a high titre was highly effective in reducing the population of pathogenic bacteria in the digestive tract, in contrast with long-term application of a lower titre.

A positive effect of phage therapy was also observed in combating horizontal infections induced by strains of S. Gallinarum in flocks of laying hens. Treatment using bacteriophages as a feed additive for chickens having contact with infected individuals led to a mortality rate of only 5%, as compared to 30% in the group that did not receive phage therapy [32].

The effectiveness of phage therapy may also depend on the individual antibacterial properties of a given bacteriophage and on the adaptive mechanisms of the bacteria. A study by Andreatti Filho et al. [30] showed that the use of selected bacteriophages in an orally administered cocktail to prevent colonization by S. Enteritidis strains in poultry was only effective for a short time (about 48 h), with no long-term protective effect, which was partly due to acquisition of resistance to the bacteriophage by the bacteria. All treatments resulted in a significant, 6-log reduction in Salmonella Enteritidis strains recovered from caecal tonsils at 24 h as compared to untreated controls, but no significant differences were observed at 48 h following treatment.

It seems promising that a wide range of lytic activity against three serovars of Salmonella – Enteritidis, Typhimurium, and Hadar – were obtained in 36-day-old broiler chicks, in which a significant reduction in the concentration of bacteria was noted following experimental infection with these serovars, by 2–4 log units [29]. The authors suggest that adjustment of treatment conditions may make it possible to use just one or two bacteriophages rather than many. In another study, Ahmadi et al. [35] demonstrated 100% efficacy in eliminating S. Enteritidis strains from the tonsils of 33-day-old Salmonella-free quails, 6 h after oral application of 100 ml of a 109 to 1010 PFU ml−1 bacteriophage suspension. It should be noted that all birds received the bacteriophage suspension for 3 days, and the therapeutic effect was noticeable within 6 h after experimental infection. The authors also confirmed that this treatment has a prophylactic effect in quails receiving 100 μl of 106 PFU ml−1 bacteriophages via oral gavage for 3 days, once every 24 h, before oral challenge with 100 μl of 1.2 × 109 CFU ml−1 S. Enteritidis. Significant prevention of colonization by S. Enteritidis strains was observed over a period of 7 days at a rate of 20% in comparison to the control (100% colonization).

Other studies suggest that bacteriophages may be used in combined treatment with other preparations, as indicated by a significant (about 80%) synergistic antibacterial effect of a commercial oral probiotic preparation applied together with a bacteriophage ‘cocktail’ of phages S2a, S9, and S11 (5.4 × 106 PFU/0.5 ml/bird) at 4, 5, and 6 days of age and at 8, 9, and 10 days of age to combat S. Typhimurium infections in broilers. The authors showed that chickens treated with a probiotic and bacteriophages showed 10 times fewer bacteria in the ileum, caecum, liver and spleen than did untreated challenged chickens. [31].

In another study, simultaneous application of three phages (MOI 103) at 108 PFU/ml/dose at 6 days of age (two daily doses) by aerosol spray and probiotics administered at 1 day of age by coarse spray, followed by oral inoculation with 2.95 × 105 CFU/ml in seven-day-old chickens, reduced Salmonella incidence and Salmonella intestinal colonization, leading to complete elimination of deaths in broiler chickens caused by infection with Salmonella Enteritidis [36]. Similar results were obtained in inhibiting horizontal infection with Salmonella following application of a bacteriophage suspension in the amount of 105 and 10 PFU/g as a feed additive for chickens challenged with 5 × 107 CFU of bacteria. Different groups of birds were treated with different titres of bacteriophage contained in the feed additive for 21 days after Salmonella Enteritidis challenge. These preventive measures significantly inhibited the replication of pathogens in the digestive tract of the chickens; however, this effect was observed mainly in chickens treated with bacteriophages at concentrations of 109 PFU/mL, which were compared only with the positive control groups [37]. Authors also suggest the occurrence the horizontal transmission of Salmonella Enteritidis strains, which was confirmed by a substantial reduction in the number of chickens treated with bacteriophages at concentrations of 107 and 109 PFU/g 1 week after treatment in comparison with untreated chickens. However, there was no significant reduction in Salmonella counts after two and 3 weeks of treatment as compared with the positive control group. And in many cases, the efficacy of phage therapy should be maximized by using a high titre of bacteriophages in order to reduce colonization by Salmonella via passive transmission.


Phage therapy has also proven to be an effective therapeutic tool in fighting pathogenic strains of Escherichia coli, particularly in preventing the development of colibacillosis, which initially develops in the respiratory tract and air sacs and then takes the form of sepsis, causing considerable mortality in poultry.

Phage suspensions applied directly to the air sac in 3-day-old birds in a range of titres from 106 to 103 PFU to treat E. coli infections substantially reduced mortality rates to 5% and 25%, respectively. Similar results were obtained after inoculation of a bacteriophage suspension in the drinking water of birds at 1 week of age (103 or 104 PFU of bacteriophages per mL) followed by air sac challenge with 103 CFU of E. coli phages. Mortality was decreased to 25% and 5%, respectively. No mortality was observed in chickens treated with 108 PFU of an E. coli bacteriophage mixture [38]. Bacteriophages have also been shown to be highly effective in treating sepsis and meningitis in newly hatched and 3-week-old chicks infected intramuscularly and intracranially with a strain of E. coli. Mortality in the untreated chicks was 100%, whereas intramuscular administration of phage R at titres of 104 and 106 PFU completely eliminated deaths in chickens in the group of treated birds. Another positive effect of the treatment was the absence of visible clinical symptoms. In the chickens intracranially infected with E. coli, application of a higher dose of the phage, at a titre of 108 PFU, fully protected the birds against the development of infection. Intramuscular application (in different muscles) of phage R at a titre of 106 PFU resulted in a lack of morbidity or mortality in all chickens. Administration of lower doses from 104 PFU of the phage after challenge with E. coli also provided significant protection, indicating that the phage had multiplied in vivo. However, the application of phages in lower doses, e.g. 102 PFU, produced no statistically significant protection against E. coli infection.

The authors also demonstrated that the bacteriophages administered to the birds intramusculary had the ability to penetrate the blood-brain barrier, and confirmed that the bacteriophages had a prophylactic effect in addition to the therapeutic effect. In 3-week-old birds effective protection against morbidity and mortality following intracranial inoculation with E. coli was obtained only after administration of 108 PFU of the phage. Only in younger birds was statistically significant protection obtained after administration of 106 PFU of the phage. Application of a suspension 1–2 days prior to experimental infection with E. coli in chicks reduced the mortality rate by 70%, as well as the intensity of the course of infection [26]. The use of bacteriophages at titres of 104–102 PFU in the form of an aerosol in chicks with symptoms of colibacillosis significantly reduced the mortality of the chicks and prevented infections in other birds. Aerosol administration of bacteriophage SPR02 at a titre of 108 PFU/mL combined with challenge with 104 CFU/mL of E. coli completely protected the birds against infection. When these phages at 104 PFU/mL were mixed with 104 CFU/mL of E. coli, mortality was significantly reduced to 35%.

The authors suggest also that similar effects preventing early development of colibacillosis in chicks are obtained by applying a bacteriophage suspension in ovo [39]. The authors also demonstrated that the effect of this kind of bacteriophage treatment is comparable to enrofloxacin treatment, and suggest that a combination of enrofloxacin and bacteriophage treatments could be efficacious and beneficial in controlling colibacillosis.

Apart from bacteriolytic activity, the effectiveness of bacteriophages is also determined by the site and route of administration of the preparation. According to Huff et al. [40] bacteriophages should be applied directly to the site of infection, which was confirmed during treatment of E. coli infections in the air sacs of chickens. Application of bacteriophages per os with drinking water proved ineffective in treating the infection and reducing clinical symptoms. When a suspension was injected directly into the air sac, an effective protective effect was obtained, manifested as the absence of clinical symptoms. This treatment significantly reduced mortality from 50 to 20% when given immediately after challenge, but had little efficacy when administered 24 or 48 h after challenge. IM injection of bacteriophages significantly reduced mortality from 53 to 17%, 46 to 10%, and 44 to 20% when given immediately, 24 h, or 48 h after challenge, respectively.

A similar effect eliminating disease symptoms in E. coli respiratory infections in poultry was obtained in broiler chickens aged 10 days to 2 weeks following repeated application of a two-phage (SPRO2 and DAF6) suspension in aerosol spray form after challenge with E. coli by injection of 104 CFU into the thoracic air sac. The authors observed the best overall protection after aerosol treatment with phage titres of 2.6 × 108 and 2.35 × 109 PFU/mL for SPR02 and DAF6, respectively. The study found a significant decrease in mortality ranging from 20% and 27% in comparison to chickens untreated with bacteriophages, but mortality was still high [40]. In the septic form of colibacillosis intramuscular application proved more effective than aerosol application, particularly in the initial phase of sepsis. In another study on broiler chicks, Huff et al. [41] demonstrated a prophylactic effect of application of a bacteriophage suspension in the form of a single intramuscular injection of two different bacteriophages (109 PFU/mL) in combination with enrofloxacin administered in drinking water immediately after E. coli challenge. Mortality decreased significantly to 15% in comparison with untreated birds challenged with E. coli (68%). The authors also demonstrated a significant synergistic protective effect in chickens that received both the bacteriophage and enrofloxacin. Because colibacillosis in poultry develops in the respiratory system, some studies suggest that bacteriophages should be applied in aerosol form as a preventive measure 1–3 days before anticipated infection, e.g. transport or transfer to a new environment [42]. The incidence of disease in 7-day old chicks treated with a phage aerosol in the first few days following experimental infection fell to under 10%, while the mortality rate in the chicks that did not receive the aerosol was 60%. A study by Oliveira et al. [43] confirmed that colibacillosis-induced high morbidity and mortality in poultry may be significantly reduced by aerosol spraying of housing systems with bacteriophage cocktails and oral administration of bacteriophages. In that study 1 ml of the phage suspension at a high titre of 1.0 × 109 PFU/ml and a lower titre of 5.0 × 107 PFU/ml of phages phi F78E, F258E and F61E was dispensed into the birds’ mouth with a syringe and by spraying directly into the beak, through a spray nozzle set to release 1 ml per fine drop scatter. Immediately after phage administration the chickens were challenged with a pathogenic E. coli suspension. The results also demonstrated a protective effect of the bacteriophages against new colonization by E.coli strains in the days following the challenge. The study confirmed the therapeutic effectiveness of one of the phages, phi F78E, administered orally and by spray at 1 × 109 PFU/mL, resulting, on average, in a 25% decrease in mortality and a 41.7% reduction in morbidity in the chickens.

The direct or aerosol administration of bacteriophages in poultry and evaluation of their therapeutic effect has been a subject of study at many research centres. A study by El-Gohary et al. [44] demonstrated that bacteriophage treatment of litter by spraying 200 mL of a bacteriophage preparation at a titre of 8 × 108 PFU/mL on the surface of 3.9 m2 pens significantly reduced the mortality of male broiler chickens (about 2–3 weeks old) with colibacillosis resulting from exposure to E. coli in the litter, even when the birds were exposed to cold stress, and furthermore reduced shedding of the pathogen among flocks.


The potential uses of phage therapy against Campylobacter bacteria may offer an alternative means of eliminating bacteria in the digestive tract of birds. This pertains in particular to infections induced by Campylobacter jejuni and C. coli, which constitute 80% of the bacteria colonizing the digestive tract in poultry. One of the first attempts to use bacterial viruses against Campylobacter bacteria was a study by Wagenaar et al. [33], in which colonization by C. jejuni was inhibited in 10-day-old chicks and adult birds, first by 2 and then by 1 log unit in broiler caeca. The Ross broiler chickens received phages by oral gavage from day 7 to 16 at different titres varying from 4 × 109 to 2 × 1010 PFU and were orally challenged with 1 × 105 CFU of C. jejuni on day 10.

The authors confirmed that phage treatment prior to bacterial challenge does not prevent but may delay bacterial colonization. However, chicks receiving phages after C. jejuni colonization showed an immediate 3-log reduction in CFU counts. It should be emphasized that the bacteria were not completely eliminated in this study, which is the main problem in the use of phage therapy for elimination of Campylobacter strains in poultry [33].

In another study, conducted at the University of Nottingham in the UK on 25-day-old chickens, following application by oral gavage of bacteriophages CP34 or CP8 isolated from the environment against C. jejuni strains HPC5 and GIIC8 obtained from birds and humans, a substantial but short-lived reduction in the number of bacteria was obtained in the intestines of infected birds, ranging from 0.5 to 5 log units. A considerable reduction in the total number of Campylobacter bacteria in the upper and lower digestive tract and in the caecum was obtained when bacteriophages were applied at a concentration of 107–9 PFU [45]. A study evaluating the effect of bacteriophages on the number of Campylobacter jejuni in the caecum in broiler chickens also confirmed a significant (P < 0.001) reduction in the total number of bacteria, to a value of 105.1 CFU/g, in comparison with chickens that were not treated with bacteriophages (mean density of bacteria 107 CFU/g) [46]. A similar reduction in the number of Campylobacter jejuni and Campylobacter coli bacteria in infected birds was obtained following application of a suspension of bacteriophage CP220 at titres of 107 and 109 PFU/ml for 5 days. A reduction in C. jejuni bacteria was observed just 48 h after injection of the phage, while in the case of C. coli a significant decrease in the number of infected birds was obtained following administration of a bacteriophage suspension with a density of 109 PFU. It should be emphasized that the percentage of birds resistant to a second Campylobacter infection was very low, at about 2% [47].

The use of a suspension of bacteriophages specific for Campylobacter jejuni and C. coli bacteria in the water or feed of broiler chickens caused a significant decrease of nearly 2 log10 CFU/g in colonization by both species of bacteria. Moreover, in contrast with earlier research, the bactericidal effect of the phages was maintained for over 7 days, enabling application of the suspension at each stage of the production cycle [48]. Preventive treatment delayed but did not prevent colonization. Levels of C. jejuni were initially 2 log units lower than in controls, and then stabilized at 1 log unit lower than in the controls.

On the other hand, the use of bacteriophages to prevent colonization by Campylobacter spp. bacteria in newly hatched broiler chicks was only partially successful. Application by oral gavage of a phage suspension with 0.4 to 2 × 1010 PFU/mL of phage 71 in 10-day-old broiler chickens initially reduced the total number of bacteria, but colonization by pathogens re-occurred within 24 h [33]. The studies cited also showed that resistance of Campylobacter spp. to particular phages was about 4%. For this reason the authors suggest creating a combination of several bacteriophages specific for Campylobacter, which in vitro research has shown to improve the effectiveness of phage therapy [49].

Clostridiosis and listeriosis

Phage therapy was shown to be effective in the case of infection of broiler chickens with anaerobic Clostridium perfringens inducing necrotic enteritis [34]. Bacterial toxins produced by this bacterium are responsible for generalization of the disease process, cause a decrease in feed intake, and inhibit growth. Oral administration to chickens of various ages of a suspension of a cocktail (INT-401) of 5 different C. perfringens phages (CPAS-7, CPAS-12, CPAS-15, CPAS-16, and CPLV-42) at titres of 105 PFU/mL, with feed or water or by oral gavage and spray application, led to a significant decrease (P ≤ 0.05) in mortality during the 0-to-42-day of experiment in comparison with the group of untreated birds. These measures also improved weight gain in the chicks. It should also be emphasized that the treatment proved more successful in reducing mortality than a formalin-inactivated vaccine containing C. perfringens alpha toxin. However, the study cited confirmed the high efficacy of the bacteriophages in controlling necrotic enteritis in poultry.

Besides whole phages, phage enzymes (endolysins and mureolytic enzymes), including murein hydrolase, merit particular attention as an additive element in combating infections induced by C. perfringens. These enzymes, binding directly to the peptidoglycans of the cell walls of Gram-positive bacteria, cause rapid lysis of these bacteria, including cells infected with bacteriophages, which accelerates their destruction. Simultaneous use of bacteriophage preparations and endolysins against Gram-positive bacteria such as Clostridium spp. and Listeria monocytogenes seems to have a highly beneficial effect. This has been confirmed in the case of use of a bacteriophage (ϕ3626) against C. perfringens, whose spectrum of lysogenic activity was at a level of 22%, while the lytic effect was 8%. In the case of combined application of bacteriophages with murein hydrolase, a lytic effect was observed against all (n = 51) tested strains of C. perfringens [50, 51]. In view of the fact that bacteriophages eliminate C. perfringens bacteria mainly by lysogeny, supplementation of phage preparations with endolysins would seem to be necessary for continuation of successful treatment.

The bactericidal effectiveness of phages has also been confirmed in fighting infections induced by Listeria monocytogenes, which like Campylobacter spp. or Salmonella is included among zoonotic pathogens inducing food poisoning in humans, with a high mortality rate of 30%. Application of bacteriophages on the surface of poultry products ready for consumption reduced the number of bacteria by 2.5 log units on a product stored at 30 °C after just 5 h. Later testing for Listeria monocytogenes in food samples kept in cold storage also yielded positive results, as the pathogen was not detected for a period of 21 days or use of a bacteriophage mixture on poultry carcasses could completely eliminated L. monocytogenes [52, 53]. Due to the risk posed by the occurrence of poultry infections induced by L. monocytogenes, as well as their increasing drug-resistance and efforts to limit the use of antibiotics, international and American health organizations are attempting to replace antibiotics with other preparations. This resulted in FDA approval on 18 August 2006 of 102-LMP™, a suspension of bacteriophages specific for L. monocytogenes, as an antibacterial agent against L. monocytogenes. This product has been estimated to successfully kill over 170 strains of Listeria spp. [54].

The main obstacles to the use of phage therapy in poultry

The full summary about using of bacteiophages in poultry’s experimental activities are included in Table 2. Despite the significant positive aspects of phage therapy, there are also some limitations in the widespread use of bacteriophages to eliminate pathogens. One of the main obstacles to elimination of bacteria from poultry is that significant numbers of phages are needed to adsorb individual host cells [50]. Some authors [38] have shown that the application of phages in lower doses, e.g. 102 PFU, provided no statistically significant protection against E. coli infection. Moreover, preventive treatment in phage therapy did not prevent colonization [48].

Table 2 Summary of studies on phage therapy in bacterial infections in poultry

In some cases a protective effect was obtained only in younger birds after high (106 PFU) doses of phage administration [26]. In many cases, the efficacy of phage therapy should be maximized by the use of a high titre of bacteriophages to reduce Salmonella colonization by passive inundation. An additional obstacle in the use of phage therapy is that colonization of chicken caeca by S. enterica serotypes Enteritidis and Typhimurium is inhibited for only 24 to 48 h after phage treatment. For this reason it seems necessary to determine the optimal timing and delivery of bacteriophages in a real-life poultry industry setting [37]. An important problem in phage therapy is that only strongly lytic phages are suitable. An area of safety concern is the potential release of toxic proteins from the lysing bacteria. In some cases, lysing bacteria inside a patient are known to release endotoxins that cause fever, and sometimes toxic shock [55].

Furthermore, the use of a defined phage or phage mixtures with largely uncharacterized genomes seems to be dangerous. Only full characterization and screening of phages can eliminate those that encode toxic proteins or proteins that allow temperate (integrative) phage behaviour. An important disadvantage in terms of safety is immune responses induced by the phage. All phages contain foreign proteins which could induce an immune response potentially reducing the effectiveness of the therapy, or even cause death as a consequence of anaphylactic shock [56, 57].

To increase the safety of bacteriophages in the elimination of pathogens, the following can be recommended: the use of only strong lytic bacteriophages, not lysogenic phages, switching to non-lysing tailocins if toxic proteins released from the bacteria become a problem; the use of rapid DNA sequencing to characterize phages used in therapy; and prescreening of patients for hyper-immune reactions to the specific phage sample prior to injection, especially in whole flocks.


The increasingly observed acquisition of antibiotic resistance by bacteria necessitates new strategies for combating drug-resistant bacteria. The results of research on bacteriophages, indicating that they can be an alternative means of eliminating pathogens posing a threat to humans and animals, justify its continuation, particularly in view of increasing drug-resistance in bacteria and restrictions on the use of antibiotics. The development of adequate phage preparations may in the future prove to be one of the most effective methods for fighting bacteria that are pathogenic for humans and animals, and will also make it possible to obtain products that are safe and free of antibiotics.