Introduction
Bacteriophages are the most abundant form of life on earth, present wherever there is a potential host – a bacterium. An important factor facilitating the acquisition and characterization of bacteriophages, in terms of their suitability for combating bacterial infections, is their common occurrence in diverse environments (e.g. wastewater, water bodies, soil, forest undergrowth, and food products). Their presence has also been confirmed in commercial sera, human vaccines, the human mouth (dental plaque and saliva), and the gastrointestinal tracts of human beings and other animals. The presence of bacteriophages is a natural phenomenon that has existed for billions of years, resulting in the balance of various bacteria in the natural environment (Batinovic et al., Reference Batinovic, Wassef, Knowler, Rice, Stanton, Rose, Tucci, Nittami, Vinh, Drummond, Sobey, Chan, Seviour, Petrovski and Franks2019).
The total number of bacteriophages on Earth has been estimated at 1032 virions, which is 10 times the number of characterized bacteria. The phage population in water bodies has been determined to range from 104 to 108 virions mL−1, while in the soil it reaches about 109 virions g−1 (Weinbauer, Reference Weinbauer2004; Wittebole et al., Reference Wittebole, De Roock and Opal2014). Currently, more than 25,000 bacteriophage nucleotide sequences have been deposited in INSDC databases (Adriaenssens et al., Reference Adriaenssens, Krupovic, Knezevic, Ackermann, Barylski, Brister, Clokie, Duffy, Dutilh, Edwards, Enault, Jang, Klumpp, Kropinski, Lavigne, Poranen, Prangishvili, Rumnieks, Sullivan, Wittmann, Oksanen, Gillis and Kuhn2017).
The mechanisms of activity of bacteriophages
Bacteriophages are characterized by specific mechanisms of action against host cells:
• Replication takes place exclusively in live bacteria that are susceptible to a given phage. The means of replication has similarities to eukaryotic viruses.
• In both lytic and lysogenic cycles, adsorption, penetration, replication of nucleic acids, formation of virions, and their release from the host cell occur.
• Phages are specifically associated with a specific bacterial strain.
• Phages can transmit new genes to microorganisms, which contributes to the genetic diversity of bacteria and the emergence of pathogens enriched with new virulence factors, such as adhesins or toxins.
• Bacteriophages show a specific affinity for individual types of bacteria.
• The specificity and spectrum of activity of phages are determined by the presence of bacterial cell surface receptors, i.e. LPS, envelopes, fimbriae, and other proteins (Weinbauer, Reference Weinbauer2004; Skurnik and Strauch, Reference Skurnik and Strauch2006; Rakhuba et al., Reference Rakhuba, Kolomiets, Szwajcer-Dey and Novik2010).
Taxonomy and classification of bacteriophages
The criteria of bacteriophage taxonomy applied by the ICTV (International Committee on Taxonomy of Viruses, EC 48, Budapest, Hungary, August 2016) are based mainly on genome type and virion morphology, including genomic and proteomic methods. Today, bacteriophages are usually classified into more than 870 species, 14 families, over 204 genera, and more than 6000 types of phages, including 6196 bacterial and 88 archaeal viruses (Ackermann and Prangishvili, Reference Ackermann and Prangishvili2012; Krupovic et al., Reference Krupovic, Dutilh, Adriaenssens, Wittmann, Vogensen, Sullivan, Rumnieks, Prangishvili, Lavigne, Kropinski, Klumpp, Gillis, Enault, Edwards, Duffy, Clokie, Barylski, Ackermann and Kuhn2016; Adriaenssens and Brister, Reference Adriaenssens and Brister2017; Adriaenssens et al., Reference Adriaenssens, Krupovic, Knezevic, Ackermann, Barylski, Brister, Clokie, Duffy, Dutilh, Edwards, Enault, Jang, Klumpp, Kropinski, Lavigne, Poranen, Prangishvili, Rumnieks, Sullivan, Wittmann, Oksanen, Gillis and Kuhn2017). However, the classification of viruses (including bacterial viruses) is still in progress, and many changes were made in 2018. Consequentially, there are now 142 families, 81 subfamilies, and about 4978 species (ICTV, 2018).
Bacteriophages can be distinguished by shape, structure, and capsid symmetry – isometric (polyhedral) and helical (spiral), nucleic acid, and interaction with the microbial host. Phages are also distinguished by size – small, medium, or large; shape – filiform or spherical; and the presence or absence of a head and/or tail. The tailed phages are a large group of viruses which account for 96% of phages. They are grouped into three families: Myoviridae, Siphoviridae, and Podoviridae (Karthik et al., Reference Karthik, Muneeswaran, Manjunathachar, Gopi, Elamurugan and Kalaiyarasu2014; Urban-Chmiel et al., Reference Urban-Chmiel, Wernicki, Stęgierska, Dec, Dudzic and Puchalski2015; Wernicki et al., Reference Wernicki, Nowaczek and Urban-Chmiel2017).
The main division and characterization of bacteriophages is based on the type of nucleic acid (DNA or RNA) and on the structure of the capsid, which is built of structural proteins. Numerous scientific reports (Karthik et al., Reference Karthik, Muneeswaran, Manjunathachar, Gopi, Elamurugan and Kalaiyarasu2014; Adriaenssens and Brister, Reference Adriaenssens and Brister2017) confirm that bacteriophages have only one type of nucleic acid and that the vast majority of them have double-stranded, or less often single-stranded, DNA. There are also species with single- or double-stranded RNA. The detailed unofficial classification of bacteriophages proposed by the ICTV, taking into account the nature of the genomic nucleic acid and virion morphology, is presented in Table 1.
Classification of bacteriophages proposed by the International Committee on Taxonomy of Viruses (ICTV), taking into account the nature of the genomic nucleic acid and virion morphology (Baj et al., Reference Baj, Markiewicz and Piekarowicz2015)
According to the classification proposed by Goyal et al. (Reference Goyal, Gerba and Bitton1987), who classified phages based on their receptors on the host, phages may be classified as follows:
• Somatic phages – with receptors present on the cell wall.
• Capsular phages – with receptors on the capsular polysaccharide.
• Appendage phages – with receptors localized on bacterial virulence factors, such as flagella, pili, or fimbriae.
According to Wittebole et al. (Reference Wittebole, De Roock and Opal2014), bacteriophages can also be classified on the basis of the specific target bacterial host, e.g. the staphylococcal phage family (Deghorain and Van Melderen, Reference Deghorain and Van Melderen2012) or the Pseudomonas phage family (Ceyssens and Lavigne, Reference Ceyssens and Lavigne2010); the environment of the phage, e.g. marine virus or land virus; and its life cycle – lytic or lysogenic, pseudo-lysogenic, or chronic infection (Ackermann, Reference Ackermann2011; Wernicki et al., Reference Wernicki, Nowaczek and Urban-Chmiel2017).
Hence there are a number of criteria for classifying bacteriophages, according to need and their possible uses in measures taken to eliminate bacteria.
The history of bacteriophages
Phages were first discovered more than 100 years ago by the English bacteriologist Frederick Twort and the French-Canadian microbiologist Felix d'Herelle (Twort, Reference Twort1915; d'Herelle, Reference d'Herelle1917). Twort demonstrated the presence of an antibacterial element with a lytic effect in cultures of micrococci, and also confirmed that the transparent substance tested could pass through filters that were able to retain larger microorganisms, such as bacteria. Twort described this material, which is not capable of growth in the absence of bacteria, as a ferment secreted by the microorganism, the reason for which was not entirely transparent. It is also worth noting that the first reports on bacteriophages had been presented by the British bacteriologist Ernest Hanbury Hankin, who as early as 1896 had discovered an unknown ‘biological suspension’ obtained from the water of the Ganges and Yamuna Rivers, which caused lysis of the cholera bacteria Vibrio cholerae (Hankin, Reference Hankin1896).
However, the first microbiologist to isolate and describe phages, and to develop the first phage therapy, was Felix d'Herelle, who is still credited by many scientists with the discovery of bacteriophages and the therapeutic implications he proposed, known as “phage therapy.” Félix d'Herelle described his observations while examining patients suffering from or cured of ‘shigellosis’ caused by infection with Shigella spp. By treating Shigella bacteria obtained from sick patients with an 18-h active filtrate from feces, d'Herelle achieved the arrest of bacterial growth and their destruction by lysis. He also demonstrated the antibacterial activity of this ‘anti-Shiga-microbe’ by applying a phage suspension in laboratory animals as an effective treatment for shigellosis, and thereby introduced the use of bacteriophages to clinical medicine. This was also a precursor to the use of intravenous phage therapy in sick patients (Wittebole et al., Reference Wittebole, De Roock and Opal2014).
Both scientists (Twort and d'Herelle) called these agents bacteriophages, and d'Herelle suggested that there was only one phage, Bacteriophagum intestinale, of which all phages were various ‘races’. However, d'Herelle emphasized that ‘the history of phage is still older than what has been documented, which is extracted from Greek word “phagein” which means “eat” – to eat or devour the bacteria’.
Due to their specificity for bacterial target hosts, bacteriophages have been used since their very discovery in various types of targeted human therapies, particularly the treatment of acute and chronic dermatological, ophthalmological, urological, oral, pediatric, otolaryngological, and surgical infections. It should be emphasized that significant therapeutic successes were achieved in the initial period of use of phage therapy, which constituted a major contribution to the development of phage therapy to treat bacterial diseases, especially in the pre-antibiotic era. According to sources from that time, the only treatment available in the 1920s and most of the 1930s was serum therapy for selected pathogens, such as pneumococci and the diphtheria bacterium, so the introduction of bacteriophages came to significantly dominate human medicine (d'Herelle, Reference d'Herelle1931; Abedon et al., Reference Abedon, Kuhl, Blasdel and Kutter2011).
According to studies on the use of bacteriophages in clinical treatments, the first article was published in Belgium by Bruynoghe and Maisin (Reference Bruynoghe and Maisin1921), who used bacteriophages to treat skin necrosis caused by staphylococci, resulting in a significant improvement in the patients’ clinical condition (i.e., reduction of pain, swelling and fever) within 48 h.
During the interwar period, the molecular structure of bacteriophages was not yet known in detail, so many scientists held the view that these microorganisms were derived from protein alone and acquired their antimicrobial properties as a result of various reactions (Northrop, Reference Northrop1938). It was not until the 1940s that the structure and shape of bacteriophages were first described and documented using an electron microscope (Rusca, Reference Rusca1940). Various types of phages were presented as photograms and their common structure was described as a non-uniform round head with a thin tail (Luria and Anderson, Reference Luria and Anderson1942). The appearance of electron microscopy, which remains a widely used technique, also enabled recognition of the stages of multiplication of bacteriophages involved in bacterial lysis, e.g. adsorption, penetration, and proliferation, and the release of daughter phages following lysis. Confirmation of the ‘viral’ nature of phages as well as the physicochemical properties of phage particles enabling their replication and lysogeny was made possible by advances in science, including the discovery of the structure of DNA and RNA molecules in the 1950s (Sankaran, Reference Sankaran2010).
Modern, rapidly advancing methods for the identification of microorganisms are to a large extent based on comprehensive genetic analysis of individual fragments of microorganisms, including bacterial viruses. This has made it possible to sequence an entire phage genome (Sanger et al., Reference Sanger, Coulson, Hong, Hill and Petersen1982) or selected subunits. This in turn has led to the possibility of restriction analysis of phages in their identification (Luria and Human, Reference Luria and Human1952; Bertani and Weigle, Reference Bertani and Weigle1953). The last decade has seen the rapid development of mass spectrometry (e.g. MALDI TOF) and proteomics, which enables highly detailed identification of bacteriophages at the genus level by means of analysis of selected amino acid fragments of protein structures, as confirmed in our previous work (Urban-Chmiel et al., Reference Urban-Chmiel, Wernicki, Wawrzykowski, Puchalski, Nowaczek, Dec, Stęgierska and Alomari2018b).
The appearance of chemotherapeutics in the 20th century, with the introduction of sulfonamides and of penicillin (discovered by Fleming in 1928) in the 1940s, resulted in a significant decline in research into the use of bacteriophages to fight bacteria. In Western Europe, phage therapies were completely eliminated from medical research, although they remained an active area of research and development in Eastern Europe, including in the republics of the Soviet Union, mainly Georgia (the Eliava Institute in Tbilisi), as well as in Poland (the Phage Therapy Unit of the Hirszfeld Institute in Wrocław) and to a lesser extent in India. It is worth noting that during the last decade, the emergence of multi-drug-resistant bacteria has led scientists to reconsider this century-old approach and to have a fresh look at phage therapy as a ‘new’ and potentially effective treatment option for difficult-to-treat bacterial pathogens (Weber-Dąbrowska et al., Reference Weber-Dąbrowska, Mulczyk and Górski2000).
Currently, in the era of increasing widespread drug resistance among microorganisms and the lack of effective methods for combating infections, phage therapies are beginning to experience a renaissance. There is practically no scientific center in the world where such research is not conducted, as confirmed by numerous publications and scientific conferences. The most important problem in combating many infections is the high multi-drug resistance of strains. This is the result of widespread and uncontrolled use of antibiotics, e.g. as growth stimulants in the form of feed additives and in treatment of bacterial infections. In human bacteria, most antibiotic resistance genes have emerged due to direct contact with strains derived from animals. For this reason, in many cases, the therapeutic effect is negligible, and the threat of infection caused by the increase in pathogens in the environment is an important factor necessitating the search for alternative methods to eliminate chemotherapeutic-resistant microorganisms.
According to numerous sources (Gardette and Tomasz, Reference Gardette and Tomasz2014; McGuinness et al., Reference McGuinness, Malachowa and DeLeo2017), methicillin-resistant S. aureus (MRSA) strains, responsible for serious nosocomial infections, have been recognized as the most dangerous type of bacteria. The significant spread of strains that are often susceptible to only one group of drugs (glycopeptides, e.g. vancomycin) is a serious problem. This phenomenon should be considered very dangerous, especially as the first vancomycin-resistant strains have already appeared in the world (including Poland), posing serious risks to the health and life of patients while causing a huge increase in health care costs (Gardette and Tomasz, Reference Gardette and Tomasz2014). Infections in the USA in 2011 caused 80,000 severe cases and 11,000 deaths. Asymptomatic colonization of the nasal cavity in the general population is estimated to range from 1.5% for MRSA to 30% for other S. aureus strains.
Phage experimental therapies in animals
The high efficacy and safety of phage therapy in comparison with antibiotics is due in part to their specificity for selected bacteria, which is manifested by the ability to infect only one species, serotype, or strain. Such a mechanism does not cause destruction of commensal gut microflora, and due to the self-replication of bacteriophages during therapy, repeated applications are often unnecessary. It is also worth noting that the mechanisms of bacterial resistance against phages and antibiotics show significant species differences (Scott et al., Reference Scott, Timms, Connerton, Carrillo, Radzum and Connerton2007a; Sultan et al., Reference Sultan, Rahman, Jan, Siddiqui, Mondal and Haq2018), Therefore, the use of phages in human medicine, veterinary medicine, or the agricultural industry does not significantly affect the susceptibility of bacteria to antibiotics used in human treatment, which is a crucial issue in the use of antibiotics in the agricultural industry. Some studies have confirmed that a single application of bacteriophages completely eliminates pathogenic bacteria, in contrast to some antibiotics, which must be administered multiple times (Lee and Harris, Reference Lee and Harris2001; Bach et al., Reference Bach, McAllister, Veira, Gannon and Holley2003; Brüssow and Kutter, Reference Brüssow, Kutter, Kutter and Sulakvelidze2005; Rivas et al., Reference Rivas, Coffey, McAuliffe, McDonnell, Burgess, Coffey, Paul Ross and Duffy2010). Another important advantage is the lack of species barrier in the antibacterial activity of phages, which means that the same bacteriophages can be used to combat infections in human and animal hosts, including pathogens such as Staphylococcus spp., various serotypes of Escherichia coli, or other species (Alomari et al., Reference Alomari, Nowaczek, Dec and Urban-Chmiel2016).
Many studies (Lee and Harris, Reference Lee and Harris2001; Sheng et al., Reference Sheng, Knecht, Kudva and Hovde2006; Wall et al., Reference Wall, Zhang, Rostagno and Ebner2010) have confirmed that bacteriophages used in targeted experimental therapies can be used to prevent and treat various bacterial infections in livestock. A reduction of up to 99% or in some cases even 100% in bacterial pathogens, including zoonotic pathogens, has been shown to significantly improve clinical outcomes in many experimental treatments of infections in cattle or pigs.
As bacteriophages are ubiquitous in the environment, their use in veterinary medicine or animal and plant production is one of the most environmentally friendly antibacterial treatments available today. This means that they have no negative effect on the environment, as in the case of antibiotics or other chemotherapeutics.
Phages have several important advantages over antibiotics that make their use in various livestock industries potentially very appealing. Some examples of experimental bacteriophage therapy include treatments for Salmonella and E. coli infections in mice, poultry, calves, piglets, and lambs; for Clostridium spp. and Pseudomonas aeruginosa infections in mice or other laboratory animals, such as hamsters and rats; and for Staphylococcus aureus infections in mice, cows, and other livestock. The advantages and potential disadvantages of the use of phage therapies are presented in Table 2.
Advantages and disadvantages of phage therapies.
In the case of phage therapy in livestock intended for consumption, many experiments have dealt with combating infections caused by zoonotic microorganisms that pose a threat to human health, particularly pathogenic strains of E. coli, Salmonella spp., Campylobacter spp., and Listeria spp., which are foodborne pathogens. As cattle, swine, sheep, goats, and poultry are raised as livestock for food, these bacteria have a significant impact on the safety of health and life of people. Proposals for replacing antibiotics as supplements result in part from current legal regulations in the European Union prohibiting the routine use of antibiotics in farm animals (Dibner and Richards, Reference Dibner and Richards2005) and limiting the chemical treatment of carcasses during processing (Atterbury, Reference Atterbury2009).
The use of bacteriophages to treat human infections has had a high success rate (about 85% or even more), especially in the case of mixed infections caused mainly by S. aureus, Klebsiella, E. coli, Proteus, Pseudomonas, Enterobacter, and vancomycin-resistant Enterococcus (Smith and Huggins, Reference Smith and Huggins1983; Smith et al., Reference Smith, Huggins and Shaw1987a, Reference Smith, Huggins and Shaw1987b; Weber-Dąbrowska et al., Reference Weber-Dąbrowska, Mulczyk and Górski2000; Morozova et al., Reference Morozova, Vlassov and Tikunova2018). In experimental treatments in livestock, bacteriophages have proven to be an effective tool in combating diseases in poultry, cattle, sheep, pigs, and fish. For example, the effectiveness of phage therapy has been confirmed in necrotic enteritis induced by anaerobic bacteria of the species Clostridium perfringens in poultry. The types and effectiveness of phage therapies used in poultry have been the subject of numerous publications (Loc Carrillo et al., Reference Loc Carrillo, Atterbury, El-Shibiny, Connerton, Dillon, Scott and Connerton2005; Scott et al., Reference Scott, Timms, Connerton, El-Shibiny and Connerton2007b; Atterbury, Reference Atterbury2009). Phage preparations in the form of cocktails have been used in poultry in experimental therapies against infections caused by pathogens such as Salmonella spp., E. coli, and Campylobacter spp. A detailed description of the use of bacteriophages to control bacterial infections in poultry, including zoonotic infections, has previously been described in our review article (Wernicki et al., Reference Wernicki, Nowaczek and Urban-Chmiel2017).
Research on phage therapy in large and medium-sized farm animals, such as cattle, pigs, sheep, and goats, plays an important role. The use of bacteriophages in cattle and other livestock species has proven an effective tool in reducing bacterial colonization in the course of chronic skin ulcers, caused mainly by staphylococci and streptococci, as well as respiratory diseases. In many cases, the number of bacteria has been limited to 2–4 log10 CFU, which was reflected in the course of the disease process as mitigation of disease symptoms (Tiwari et al., Reference Tiwari, Dhama, Chakraborty, Kumar, Rahal and Kapoor2014).
In ruminants, experimental phage therapies have been tested to combat infections in newborn calves and lambs, caused mainly by enterotoxigenic E. coli strains. The studies have confirmed the effectiveness of these therapies, based on mitigation of disease symptoms (diarrhea and fever) and a reduction in mortality ranging from 15% to about 67%. In addition, the phages remained in the gut of the animals for as long as pathogenic E. coli strains were present.
Experimental phage therapy in ruminants
Because cattle and other ruminants are the main reservoirs of pathogenic and zoonotic strains of E. coli, including O157:H7, a great deal of research concerns the use of bacteriophage treatment to eliminate these pathogens (Goodridge and Bisha, Reference Goodridge and Bisha2011).
The effectiveness of phage therapy against infections caused by enterotoxigenic E. coli strains in newborn calves has been varied, and researchers (Johnson et al., Reference Johnson, Gyles, Huff, Ojha, Huff, Rath and Donoghue2008) have shown that it depends on a number of factors, including the following:
• the experimental design of the infection
• the form of phage application
• the quantitative and qualitative composition of the dose of bacteriophages
In an early study, Smith and Huggins (Reference Smith and Huggins1983) used a phage cocktail containing a mixture of two phages, B44/1 and B44/2, at a titer of 1011 PFU mL−1, against the enterotoxigenic E. coli strain O9:K30.99, inducing enteritis in calves. The authors demonstrated high (nearly 93%) efficacy of the experimental treatment in calves that were not fed colostrum but treated with phages, even when clinical symptoms were present. The results confirmed that a phage cocktail can significantly reduce morbidity and mortality, even when applied in the case of significant clinical symptoms.
In research by Callaway et al. (Reference Callaway, Edrington, Brabban, Anderson, Rossman, Engler, Carr, Genovese, Keen, Looper, Kutter and Nisbet2008), oral application of a phage cocktail obtained for an E. coli reference strain, i.e. O157:H7 strain 933 (ATCC 43895), resulted in a significant reduction in bacteria in individual segments of the gastrointestinal tract: the rectum, caecum, and, in two cases, the rumen. The number of pathogenic bacteria isolated from animals after application of the phage cocktail ranged from 102 to 103 PFU g−1 of caecal and rectal contents in all samples tested, but only in two samples in the case of the rumen.
A significant reduction in strains of this human pathogenic serotype was also observed by Sheng et al. (Reference Sheng, Knecht, Kudva and Hovde2006) following oral administration of a bacteriophage suspension to young calves. The authors observed a significant reduction in colonization up to complete elimination of enterotoxigenic E. coli strains of serotype O157:H7 from the gastrointestinal tract up to day 16 after application.
High efficacy in reducing diarrhea and mortality has been obtained after a single per os administration of a phage mixture at a titer of 105 PFU mL−1 before or at the onset of diarrhea and simultaneous infection per os with various pathogenic strains of E. coli. Different doses of bacteriophages at titers of 102 and 105 PFU mL−1 in the period from 6 h to 10 min before challenge and up to 10–12 h after challenge resulted in a reduction in disease symptoms (fever and diarrhea). Repeated administration of bacteriophages with milk or colostrum at 105 PFU mL−1 resulted in high antibacterial efficacy in the case of administration of phages from 4 to 10–12 h after infection. The authors confirmed high phage titers of 1011 PFU mL−1 in vivo just 5 h after application, and they also reported that the application of small doses of phages with titers up to 102 PFU mL−1 immediately after the onset of diarrhea significantly alleviated disease symptoms (Brüssow and Kutter, Reference Brüssow, Kutter, Kutter and Sulakvelidze2005). Based on the diverse results obtained in experimental phage therapies in calves, it has been hypothesized that specific doses of phages can be used to ‘control the course of the disease’, because diarrhea was prevented even when the suspension was administered up to 8 h after experimental infection of calves with enterotoxigenic E. coli, and this was correlated with a simultaneous decrease in the number of pathogens.
An important element in phage therapies is their capacity for self-replication in target cells, and thus in the body of the infected individual. Waddell et al. (Reference Waddell, Mazzocco, Johnson, Pacan, Campbell, Perets, MacKinnon, Holtslander, Pope and Gyles2000) treated weaned 7- to 8-week-old calves with a six-phage cocktail up to 7 days before challenge with E. coli O157:H7 and obtained a significant increase in the number of phages excreted in feces after day 8 of application and a reduction in the number of E. coli excreted by the animals in comparison to the control animals. The increase in the number of bacteriophages was determined to be the result of their replication in bacterial cells and their release into the intestinal lumen.
Research conducted by Chase et al. (Reference Chase, Kalchayanand and Goodridge2005), in which the authors used a cocktail of 37 phages specific to E. coli O157:H7 strains in weaned Black Angus calves aged 4–6 months, showed a significant reduction in the bacterial concentration up to 16 or 24 h, depending on the number of applications. Moreover, the E. coli O157:H7 strains did not acquire resistance to any of the 37 bacteriophages used in the experiment, even after re-infection, which should be considered a highly beneficial therapeutic effect.
An important unfavorable phenomenon is the limited duration of an effective antibacterial titer of bacteriophages in ruminants, which is significantly linked to the sensitivity of bacteriophages to the gut environment. The viability of bacteriophages administered per os can significantly decrease due to the acidic environment of gastric acid, the presence of rumen microflora, and the activity of enzymes and other digestive compounds, such as bile (Goodridge and Bisha, Reference Goodridge and Bisha2011).
The effectiveness of phage therapies in calves in their first week of life following experimental infection with various ETEC strains has also been found to be determined by physiological factors in the newborn, such as the inactivating effect of gastric pH ≤ 3 and a body temperature raised to ≥40 °C due to fever, which may have a significant impact on bacteriophage virulence (Smith et al., Reference Smith, Huggins and Shaw1987b).
Some bacteriophages cannot survive in an environment with pH 2–3 or 7, which significantly reduces their titer, but sensitivity to specific pH values may also depend on the type of bacteriophage. Some research confirms (Dąbrowska et al., Reference Dąbrowska, Switała-Jelen, Opolski, Weber-Dąbrowska and Górski2005) that certain bacteriophages have a high survival rate in a pH range from 2 to 7. Furthermore, the problem of inactivation of phages by low gastric pH can be resolved by administering the phage suspension together with milk or directly after the calf feeds on milk, as confirmed by Barrow et al. (Reference Barrow, Lovell and Berchieri1998). The administration of substances neutralizing gastric acid, mainly saline solutions, immediately prior to administration of the bacteriophage suspension also appears to be an important element of effective phage therapy.
Taking into account the above assumptions, special attention has been paid to the possibility of rectal application of a phage suspension in order to bypass gastric juice and rumen microflora (Bielke et al., Reference Bielke, Tellez and Hargis2012). Research by Sheng et al. (Reference Sheng, Knecht, Kudva and Hovde2006) based on previous in vitro experiments (Naylor et al., Reference Naylor, Low, Besser, Mahajan, Gunn, Pearce, McKendrick, Smith and Gally2003) has shown that application of a KH1 and SH1 phage cocktail (1010 PFU mL−1) to young beef cattle in the form of a suspension per rectum with simultaneous administration of the same lytic phages (106 PFU mL−1) with drinking water caused a significant reduction in Shiga toxin-producing strains of E. coli O157:H7 in experimentally infected calves up to the detectable limit in most of the calves. However, this procedure did not completely eliminate pathogens from the herd.
The combination of various methods of bacteriophage application in the form of a suspension in cattle to eliminate pathogenic E. coli O157:H7 strains has also been the subject of research presented by Rozema et al. (Reference Rozema, Stephens, Bach, Okine, Johnson, Stanford and McAllister2009). The authors also used simultaneous oral and rectal administration of a 1011 PFU mL−1 suspension of phages specific for nalidixic acid-resistant strains of E. coli O157:H7. However, the treatment did not completely eliminate pathogens from the body after experimental infection of the animals. Despite the reduction observed in the number of excreted E. coli bacteria, the values were not statistically significant compared to animals not treated with phages. Moreover, fecal excretion of bacteriophages specific to the E. coli O157:H7 strain used in the study was also observed in the young beef cattle from the control group, which the authors suggest may have been due to acquisition of phages per os from the farm environment.
Similar results in the elimination of pathogenic E. coli strains responsible for diarrhea in newborn calves have also been observed in our own research (Urban-Chmiel et al., Reference Urban-Chmiel, Alomari, Dec, Nowaczek, Stęgierska, Puchalski, Wernicki and Kowalski2018a). Significant improvement in the health of newborn calves with symptoms of E. coli-induced diarrhea was achieved following six rectal applications (over 5 days) of a suppository containing a mixture of 107–109 PFU ml−1 bacteriophages specific for pathogenic E. coli, in combination with probiotic strains isolated from cattle (patent application no. P.424314). The results indicated a significant therapeutic effect of the experimental suppositories, manifested by a decrease in rectal temperature and a reduction or complete elimination of diarrhea within 24 or 48 h after the first application of phages. A preventive effect of the experimental treatment was confirmed as well, manifested as stimulation of specific and non-specific mechanisms of the humoral immune response. It should also be emphasized that no pathogenic strains of E. coli were found in the calves for 3 weeks after application of suppositories.
Another example of a means of controlling infections in calves involves spraying of a bacteriophage suspension in the form of an aerosol cocktail to eliminate microbes. The use of phages obtained from uncleaned rooms where calves are kept (bedding, remains of feed, or feces) has also been found to result in highly effective protection in calves infected 3 h after being placed in the rooms. When a bacteriophage suspension was sprayed on litter in the amount of 105–109 m−2 immediately before infection and up to 6 h before infection, bacteria were completely eliminated within 10 days after treatment. Spraying of a bacteriophage suspension in an aerosol form on the mucous membranes in the initial and terminal sections of the alimentary tract of calves within 24 h of the onset of diarrhea completely eliminated clinical symptoms within the next 20 h. Moreover, a high concentration of bacteriophages was found to persist until the end of infection, i.e. until E. coli was no longer isolated from the gastrointestinal tract, and sharply decreased after the animals had recovered, which resulted in protection of calves against diarrhea (Smith and Huggins, Reference Smith and Huggins1983).
According to Sheldon et al. (Reference Sheldon, Lewis, LeBlanc and Gilbert2006) and Machado et al. (Reference Machado, Bicalho, Pereira, Caixeta, Bittar, Oikonomou, Gilbert and Bicalho2012), bacteriophages may also have beneficial effects in reducing the incidence of uterine infections induced by E. coli. However, Meira et al. (Reference Meira, Rossi, Teixeira, Kaçar, Oikonomou, Gregory and Bicalho2013) were unsuccessful in treating cows with postpartum metritis caused by mixed flora, mainly pathogenic strains of E. coli, by intravaginal administration of a cocktail of 10 different phages at a titer of 109 PFU mL−1, specific for E. coli and without taking into account other etiological agents. Despite the reduction in the number of E. coli bacteria, there was no improvement in the health of the animals, whose disease symptoms were not alleviated. Furthermore, no preventive effect was obtained in the form of a reduction in the incidence of metritis. In addition, the incidence of retained placenta increased in cows after parturition, which the authors suggest may have been due to suppression of localized cellular immune mechanisms, including inhibition of neutrophil migration, phagocytosis, and oxidative activity. In addition, the authors indicate that the problem of metritis in dairy cows is very often the result of mixed infections induced by pathogenic strains of species such as E. coli, Trueperella pyogenes, and Fusobacterium necrophorum, which makes the elimination of infections particularly difficult. A similar situation has been observed in attempts to combat subclinical mastitis caused by S. aureus in dairy cows. Five intramammary applications of a suspension of bacteriophage K at 1011 PFU mL−1 cured only about 16.7% of cases, and this percentage was not statistically significant compared to the control group. The authors suggested that such a low success rate may have been due to the application of the phage suspension during lactation, which was linked to the activity of enzymes contained in the milk that inactivated the bacteriophages (Gill et al., Reference Gill, Pacan, Carson, Leslie, Griffiths and Sabour2006a, Reference Gill, Sabour, Leslie and Griffiths2006b).
Another example of the use of bacteriophages to combat bacterial infections in cattle was the development of a preparation using phage enzymes in combination with specific bacteriophages. This research resulted in a project to combat skin and mucous membrane infections caused by F. necrophorum in beef cattle (patent application no. WO 2004064732 A2).
In the case of experimental treatments in sheep, most research has concerned the control of infections caused by pathogenic strains of E. coli. For example, Raya et al. (Reference Raya, Varey, Cot, Dyen, Callaway, Edrington, Kutter and Brabban2006) treated crossbred sheep with a single oral gavage application of a CEV1 phage suspension at ~1011 PFU mL−1 3 days after experimental infection with pathogenic E. coli O157:H7 EDL 933 (1010 CFU per sheep), and reported a significant reduction in pathogenic strains from individual segments of the gastrointestinal tract (rumen, caecum, and rectum) 2 days after application of the phages. In another study (Smith and Huggins, Reference Smith and Huggins1983), application of a phage suspension to lambs 8 h after they were infected with an O8:K85,99 enteropathogenic strain of E. coli S13 reduced the numbers of pathogenic bacteria in the alimentary tract during the first 24 h after application. According to the authors, the experimental procedure also had a ‘beneficial ameliorating effect’ on the course of the disease.
On the other hand, a study by Bach et al. (Reference Bach, McAllister, Veira, Gannon and Holley2003), following oral administration in sheep a single phage (DC 22) at a titer of 105 PFU mL−1, apart from a reduction in the number of E. coli O157:H7 up to 13 days after phage application, found no significant effect on fecal excretion of E. coli O157:H7 on successive days of the experiment (as of day 30). The authors suggest that this was probably due to non-specific binding of phages with food particles and other waste present in the rumen and gastrointestinal tract, which could ultimately have limited their effectiveness. Other authors (Sheng et al., Reference Sheng, Knecht, Kudva and Hovde2006) have found that four per os applications of a suspension of phage KH1 specific for E. coli ATCC 43894 at 1.3 × 1011 PFU mL−1 in 7-month-old sheep caused no significant reduction in these E. coli strains in the gut.
Phage therapy in pigs
In the case of pigs, experimental applications of bacteriophages to eliminate foodborne pathogens have mostly been focused on controlling infections caused by pathogenic strains of E. coli and Salmonella spp.
The results of experimental phage therapies used in pigs to combat infections caused by pathogenic strains of E. coli have also been promising in many cases. For example, in an early experiment carried out by Smith and Huggins (Reference Smith and Huggins1983), oral application of a cocktail of two phages, P433/1 and P433/2, or phage P433/1 alone in piglets with diarrhea caused by pathogenic E. coli P433 O20:K101, 987P significantly reduced clinical signs of diarrhea and the numbers of E. coli strains excreted by the animals.
In other experiments (Lee and Harris, Reference Lee and Harris2001), simultaneous oral and intramuscular application of phage Felix 01 specific for pathogenic Salmonella Typhimurium strains at 1010 PFU mL−1 in 3-week-old piglets caused a significant reduction in the bacteria in the tonsils and caecum. In contrast, one of these author's previous study in 2000, assessing the effectiveness of a cocktail containing 26 phages specific to Salmonella spp. strains, had shown no significant reduction of bacteria in animals treated with the phage cocktail (Harris, Reference Harris2000).
In another study, two applications of a suspension of two phages targeting Salmonella enterica strains at 3 × 109 PFU mL−1 at 24 and 48 h after challenge with S. enterica serotype Typhimurium caused a reduction in bacteria of >1.4 log10 CFU in the caecum, but a significant (p < 0.05) reduction in the pathogens was observed only in the rectum (Callaway et al., Reference Callaway, Edrington, Brabban, Kutter, Karriker, Stahl, Wagstrom, Anderson, Poole, Genovese, Krueger, Harvey and Nisbet2011). These authors also emphasized that several phages should be combined in the form of a cocktail and that several applications were necessary to exclude the potential risk of resistance in the pathogens and to prolong their exposure to phages.
The research results indicate that phage therapies directed against bacteria of the family Enterobacteriaceae, particularly pathogenic strains of E. coli, have proven to be the most effective in eliminating pathogens. For example, a study on the elimination of infections caused by pathogenic strains of E. coli in piglets using phage T4 of the Myoviridae family at 105 PFU mL−1 achieved up to 100% protection against infection. The optimal concentration of bacteriophages guaranteeing complete protection in 1-month-old piglets against experimental infection with E. coli O157:H7 strains was found to be 109 PFU phages, applied three times (Skoblikow and Zimin, Reference Skoblikow and Zimin2013), but the authors suggest the need to individually adjust the concentration of bacteriophages and the individual therapy regimen.
Dietary supplementation with phage cocktails specific for mixed pathogens such as Salmonella spp., E. coli, S. aureus, and C. perfringens as an alternative to antibiotic growth stimulants has also been found to significantly improve growth performance in growing pigs (Kim et al., Reference Kim, Ingale, Kim, Lee, Lee, Kwon and Chae2014; Svircev et al., Reference Svircev, Roach and Castle2018). For example, in a study by Gebru et al. (Reference Gebru, Lee, Son, Yang, Shin, Kim, Kim and Park2010), in which weaned piglets were given a feed supplement of bacteriophages specific for S. Typhimurium at 3 × 109 PFU kg−1 of feed; Lactobacillus plantarum CJLP56 at 6.5 × 108 CFU kg−1 of feed (LP); 0.2% microencapsulated organic acids; or 5% fermented soybean meal. Bacteriophage supplementation together with probiotic strains was carried out for 2 weeks before and 2 weeks after oral challenge with S. enterica serotype Typhimurium. The results confirmed that a diet with the phage + probiotic supplement had a similar beneficial effect on growing pigs as an antibiotic-supplemented diet, especially after bacterial challenge.
In other research conducted in weaned piglets aged 3–4 weeks old, administration of an anti-Salmonella phage cocktail at the time of inoculation with S. enterica serotype Typhimurium significantly reduced Salmonella colonization by 2- to 3-log in the tonsils, ileum, and caecum, which was 99.0–99.9% of pathogens. The phage cocktail also showed lytic activity in vitro against Salmonella strains not belonging to the Typhimurium serotype. These include the Dublin, Enteriditis, Indiana, Kentucky, Litchfield, and Schwarzengrund serotypes of S. enterica (Wall et al., Reference Wall, Zhang, Rostagno and Ebner2010). The results reported by the authors confirm that bacteriophages have a broad spectrum of activity against microorganisms within one species but belonging to different serotypes. Another study by the same authors demonstrated that administration of a phage cocktail to young piglets immediately after experimental challenge with S. enterica serotype Typhimurium reduced bacterial counts to undetectable limits (by up to 95% in the tonsils and ileum, and up to 80% in the caecum).
Bacteriophages have also been observed to have antibacterial effects on Salmonella infection in weaned pigs experimentally challenged with S. Typhimurium. After treatment with a bacteriophage cocktail C containing eight phages (SEP-1, SGP-1, STP-1, SS3eP-1, STP-2, SChP-1, SAP-1, and SAP-2) with titers ≥109 PFU ml−1) (Seo et al., Reference Seo, Song, Lee, Kim, Jeong, Moon, Son, Kang, Cho, Jung and Kim2018), the authors observed lytic activity against 100% of Salmonella ATCC 14028 reference strains and 92.5% of field isolates. The study confirmed that a bacteriophage cocktail is more effective than a single bacteriophage in controlling bacterial infections in pigs.
A summary of studies with experimental phage treatments in livestock and their results is shown in Table 3.
Summary of studies of experimental phage treatment in livestock
Commercial phage products in livestock production
Bacteriophages as components of commercial products are currently finding application in the elimination of pathogens from food products of animal origin (meat and meat products, milk and dairy products) or plant origin (fruits and vegetables). Most of these preparations have been officially approved in the USA, Canada, Israel, Australia, and some European countries, such as Sweden, Switzerland, or the Netherlands (BAG, Bundesamt für Gesundheit; CFR, Code of Federal Regulations; FSIS, Food Safety and Inspection Service; GRN, GRAS Notice; European Food Safety Authority EFSA; Standards Australia New Zealand FSANZ) (Moye et al., Reference Moye, Woolston and Sulakvelidze2018). It should be emphasized that the number of positive decisions around the world regarding the marketing authorization of phage preparations as substances generally recognized as safe (GRAS) is still rising, which is significantly linked to restrictions on the use of antibiotics in animal production.
For example, the United States Food and Drug Administration (FDA) has approved three phage preparations (ListShield™″, EcoShield™, and SalmoFresh™) as effective products for reducing bacterial contamination of various foods.
In addition, food safety guidelines in the USA recognize several phage preparations as safe and suitable ingredients for use in the production of meat, poultry, and egg products. For example, FSIS Directive 7120.1 permits the use of phages in livestock prior to slaughter (e.g. phages specific for pathogenic strains of E. coli O157:H7 for use on beef hides) and in foods (e.g. phages specific for Salmonella on poultry or meat products derived from livestock).
Other products, such as PhageGuard S™, containing phages specific to pathogenic strains of Salmonella spp. and E. coli O157:H7, have been recommended in Israel, Switzerland, and Canada. A detailed list of commercial phage preparations with their recommendations as products GRAS and approved for use in food production is presented in Table 4.
Selected commercial phage preparations with a list of administrative authorities allowing their use in veterinary medicine and food production (Moye et al., Reference Moye, Woolston and Sulakvelidze2018 with our own modification)
Conclusions
Summing up the scope of knowledge on the use of bacteriophage preparations, it should be emphasized that it is useful to test experimental therapies in animals to treat bacterial infections caused by antibiotic-resistant microorganisms, as indicated by the therapeutic success that has already been observed in combating selected infections. This is also evidenced by a significant increase in approvals and registrations of commercial phage preparations for controlling pathogens occurring in the food of animal and vegetable origin.
Author contributions
M.D. and A.W. contributed to the collection and revision of the literatures and wrote the manuscript. R.U.C. was involved in the concept of manuscript, collection and revision of the literatures, and wrote the manuscript.
Conflict of interest
The authors have declared no conflicts of interest for this article.
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