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The Use of Bacteriophages in Veterinary Therapy

  • Robert J. AtterburyEmail author
  • Paul A. Barrow
Living reference work entry

Abstract

There is a long history of using phage to treat infections in animals; the first examples of which occurred soon after the discovery of phage by Twort and d’Herelle over a century ago. Many of the earlier phage therapy experiments in animals focused on either demonstrating the efficacy of this approach for treating animal diseases directly (e.g., fowl typhoid) and/or using animals as models of human disease (e.g., plague, meningitis, dysentery), to varying degrees of success. The pioneering work of Williams Smith and colleagues in this field in the 1980s spearheaded a revival of phage therapy investigations in the West which has continued to gather pace to the present day. The majority of recent phage therapy trials in animals have focused on farm/food animals rather than companion animals, primarily because of economic, regulatory, and logistical reasons. Technological advancements in the biosciences in areas such as genetics, immunology, and computational biology have allowed further refinement of phage therapy experiments in animals, particularly with respect to selection of effective candidates for therapeutic trials. The use of phage to treat animal diseases is yet to be widespread, even in countries such as the United States, where phage products have been approved for this purpose. However, the growth of antimicrobial resistance in both human and animal pathogens may yet overcome barriers to using them more extensively.

Introduction

Infectious diseases of animals can be subdivided into those which have mainly economic impacts and those which are zoonotic, i.e., transmissible from animals to humans. The most significant economic impacts occur in the livestock sector and may include the direct costs of control and prevention measures, export bans, reduced product quality, litigation, market disruption (e.g., consumer fears and supply shortages), and effects beyond the livestock industry (e.g., wildlife and tourism). These costs may be compounded if the livestock disease is also zoonotic. Approximately 75% of new diseases that have affected humans over the past 10 years have originated from animals or products of animal origin. The most common bacterial pathogens associated with food animals are Campylobacter, Salmonella, and Escherichia coli.

Respiratory infections are prevalent in young animals where levels of innate immunity are lower than in adults. Gastrointestinal infections result in enteritis, usually with diarrhea, affecting neonatal animals and those in the immediate postweaning period. The pathogens most frequently implicated in such infections include Escherichia coli, Salmonella, and Campylobacter. However, a range of parasites and enteric viruses also may be involved, either individually or as part of a mixed infection. Mixed infections are common, with viral infections predisposing to secondary bacterial infections; the latter frequently caused by members of the Pasteurellaceae (e.g., Pasteurella and Mannheimia), and others such as Ornithobacterium, Actinobacillus, Haemophilus, Streptococcus, and Staphylococcus. Septicemias often arise from respiratory infections and generally involve the same organisms. Added to this list are the mycobacteria, which produce tuberculosis in a variety of livestock species.

Many of these infections are currently addressed through a combination of biosecurity measures, vaccination, and chemotherapy. Biosecurity may include automatic disinfectant sprays at farm entrances, dedicated protective clothing for individual animal houses, anterooms and step over barriers, increased rodent trapping, and preventing the access of wildlife to feed stores. The use of antimicrobial chemotherapy in food animals is becoming increasingly difficult to justify in the West. This is due partly to the increase in antimicrobial resistance, and also pressure from consumers and regulatory bodies. Improved welfare, biosecurity and use of vaccines may all help to reduce our reliance on antibiotics. However, significant improvements in these areas are likely to be expensive; and vaccines may not address acute infections which require immediate treatment. Antimicrobial resistance is becoming a major issue, which international organizations including the World Health Organization have recognized requires a multifactorial approach, including the use of bacteriophages (WHO 2014; Reardon 2017).

Bacteria, which are resistant to multiple antimicrobials, are either well-known pathogens which have acquired resistance through mutation or horizontal gene transfer (e.g., E. coli and Salmonella) or opportunistic agents which are unsusceptible to many antibiotics (e.g., Pseudomonas aeruginosa and Acinetobacter baumanii). Many of these pathogens are zoonotic and foodborne; circulating between populations of humans and domestic/wild animals, as well as the wider environment. Efforts to reduce antimicrobial use in veterinary medicine do not necessarily lead to a reduction in resistance. Some resistance mechanisms carry little to no fitness cost (Luangtongkum et al. 2009), and in some cases may confer a selective advantage. For example, fluoroquinolone resistant Campylobacter isolates have been found to outcompete sensitive isolates when colonizing chickens in the absence of antibiotics (Luo et al. 2005). Mortality rates in human patients infected with multidrug-resistant Salmonella Typhimurium strains have been found to be at least twice that of people infected with sensitive strains (Helms et al. 2002). Moreover, the migration from antibiotic “growth promoters” in animal feed, at least in many countries in the West, has been accompanied by an increase in the use of heavy metals (chiefly Zn and Cu salts) as a substitute. This is problematic as genetic linkages between heavy metal and antibiotic resistance determinants have been found in animal and environmental studies, as well as in the laboratory, and heavy metals persist in the farm environment for extended periods of time (Wales and Davies 2015).

This chapter discusses the use of bacteriophages to control both veterinary and zoonotic pathogens in animals, i.e., those affecting only animals and those which also can affect humans. There are few examples of phages targeting veterinary pathogens which have reached field application. As such, this review will be an appraisal of experimental veterinary applications of phage therapy and the potential regulatory issues, which may affect their use in livestock and the food sector. The use of bacteriophage lysins for infection control is covered in a separate chapter (see chapter “Enzybiotics: Endolysins and Bacteriocins”).

History of Bacteriophages in Veterinary Use

Bacteriophages were discovered independently by Twort (1915) and d’Herelle (1917) (Duckworth 1976). The first example of veterinary phage use was by d’Herelle himself, in the experimental treatment of fowl typhoid (Salmonella Gallinarum) in Eastern France; initially in a pilot study with six chickens, then in a much larger trial with 100 chickens. Both trials were successful, with 95–100% of phage-treated animals surviving compared with 0–25% of untreated control birds (d’Herelle 1926). Pyle (1926) used phage to treat a similar systemic infection in chickens caused by Bacterium (Salmonella) Pullorum. The results of these experiments were more equivocal than for d’Herelle’s. Phage treatment appeared to delay rather than prevent death of the birds, regardless of the administration route (oral or intramuscular) and despite clear evidence that the phage used had a broad host range and lysed in vitro cultures of S. Pullorum.

Perhaps unsurprisingly, veterinary phage therapy experiments were often used as a surrogate to determine the efficacy of this approach for human infections. Such work included Topley et al. (1925a, b) who treated systemic Salmonella infections in mice with oral and intraperitoneal phage administration, largely unsuccessfully. Larkum (1926) found that bacteriophage could reduce E. coli populations in experimentally infected bladders of rabbits (although urine appeared to inhibit the action of the phages). Compton (1930) used groups of mice as a model of plague (Bacillus [Yersinia] pestis) and found that phage treatment (subcutaneous injection) significantly reduced mortality of the mice (67% survival vs 0% in the untreated control group). However, bacteriophage could not be recovered from phage-treated animals. Moreover, a similar percentage of mice in the phage-treated group could be protected by injection of formalin-inactivated preparations, suggesting active phage replication was not responsible for this outcome, and that protection may have been mediated by immunomodulatory levels of bacterial lysis components in the preparation.

Progress in phage therapy was hampered in the subsequent decades due to a poor understanding of bacterial pathogenesis and phage–host interactions, and was compounded by the absence of good animal models of disease. Regrettably, this led to a succession of poorly designed and executed experiments and field trials. Such mistakes included selecting poor phage candidates, which failed to lyse cultures fully in vitro (Topley et al. 1925a), and the simultaneous administration of the bacterial inoculum with the phage (Walker 1929), which essentially replicated an in vitro interaction between phage and host. After the advent of antibiotic therapy in the West in the 1940s–1950s, the idea of using phages largely became discredited (Pollitzer 1959). It was only during the 1980s that rigorously controlled phage therapy experiments were performed (Smith and Huggins 1982, 1983; Smith et al. 1987). These, together with advances in the fields of molecular biology, genomics, phage biology, and animal models of disease, have enabled a more objective reappraisal of phage therapy to be undertaken (see Discovery of Bacteriophages and the Historical Context).

Systemic Escherichia coli Infections

E. coli strains expressing the K1 surface antigen (K1+) frequently cause septicemia in chickens, usually after viral infections, in colostrum-deprived newborn calves and infant humans. Human sewage contains an abundance of phages, which specifically attach to the K1 antigen and can be used for identification purposes. Smith and Huggins (1982) used anti-K1 phages in vivo with the rationale that any phage-resistant mutants would be K1, and therefore less virulent. These phages were tested in mice, by intramuscular inoculation and were found to be highly effective even with a low multiplicity of infection (<1), indicating multiplication of the phage in vivo. K1+ phages were also effective against direct inoculation of E. coli into the brain, and were more effective than most of the antibiotics tested, including eight doses of streptomycin.

Similar results were obtained with experimental E. coli septicemia in chickens (Barrow et al. 1998). Protection was possible, even when the phage were given several days before the E. coli challenge, and also when phage administration was delayed until the birds were symptomatic, which resulted a 70% fall in mortality.

Other groups have evaluated the use of virulent phages to treat E. coli septicemia in chickens, but in under less-than-realistic conditions. One study involved mixing phage with the bacteria in vitro prior to inoculation into the air sac, and subsequently recorded a reduction in mortality from 85% to 35%. No effect was obtained by administration of the phage in drinking water (Huff et al. 2002). Protection was also obtained by E. coli inoculation of the air sac coupled with intramuscular phage inoculation (Huff et al. 2003). These authors also found evidence of antibodies specific against the bacteriophage in serum. Similar protective effects of phage treatment have been found under experimental conditions with mice when bacteria and phage are administered simultaneously, and by the same route (Soothill 1992; Merril et al. 1996). El-Gohary et al. (2014) found that spraying litter with phage also reduced the incidence of colibacillosis in broilers as Smith and Huggins (1983) found earlier in treating E. coli calf enteritis (see below).

Others have reported mixed results using phages against systemic E. coli infections in chickens. Oliveira et al. (2010) found good results using a cocktail of three phages against experimental infections with single spray or oral application, reducing mortality from 43% to 25%. Huff et al. (2010) found similarly good results with experimental intra-air sac inoculation of an E. coli strain with intramuscular phage inoculation, reducing mortality from 55% to 8%. However, antibodies were generated against the phage if they were introduced prior to bacterial challenge. Xie et al. (2005) also reported good phage-protective effects but no information at all was provided on the routes of administration for either the pathogen or phage, so useful interpretation of the data is impossible. Tsonos et al. (2014) found phages which were effective in vitro but which had no effect at all in vivo using intratracheal inoculation of an APEC strain and phage administered by several routes, including intratracheal inoculation.

Barrow et al. (1998) also applied the anti-K1 phage in colostrum-deprived calves, which had been given the K1+ E. coli orally. Intramuscular administration of the phage prevented death for several days. Some treated animals succumbed, it was thought, to endotoxemia from the enormous amount of LPS in the intestine resulting from the extensive colonization.

Systemic Salmonella Infections

Early experiments assessing the use of phages to treat systemic Salmonella infections produced mixed and equivocal results for the reasons indicated above. d’Herelle performed experiments with S. Gallinarum, which produces fowl typhoid in chickens of all ages; and S. Pullorum, a related serotype, which produces a similar disease in very young birds. He found that an increase in the prevalence and activity of a specific phage, among chickens exposed to infection during an epidemic, coincided with the cessation of the epidemic (d’Herelle 1926). He found that oral ingestion, or parenteral inoculation with a phage active against S. Gallinarum, protected birds challenged orally with the pathogen. He also found that birds infected with S. Gallinarum and treated with phage were resistant to subsequent infection (possibly by adaptive immunity). In some cases, no protective effect of phage therapy was observed, although this was attributed to infection produced by other pathogens. In a further experiment, 100 chickens were exposed to infection with S. Gallinarum, 20 of which were inoculated orally with a homologous phage. All of these birds survived whereas 60/80 of the remaining birds died. Most of the protection was thought to have occurred in the intestine.

A contrast was provided by Topley and colleagues (1925b). In this work, “lytic filtrate” was administered either orally or intraperitoneally to groups of 90 mice in experimental epidemics of S. Typhimurium (Bacillus aertrycke). The “lytic filtrate” used in the experiment seems not to have been characterized beyond confirming that the culture of S. Typhimurium fed to the mice was “known to be sensitive to the lysin employed.” Treatment had no beneficial effect on bacterial shedding or host mortality. However, the authors could only recover “active lysin” from 2/360 mice, suggesting that if any phage were infection present in the “lytic filtrate,” they were not able to actively replicate in this environment or were ineffective against phage-resistant subpopulations. Two experiments looking specifically at mortality resulted in either no effect or a delayed beneficial effect, which they attributed to the development of immunity.

S. Typhi infection can be lethal for mice, but only following artificial infection, which requires a high intraperitoneal bacterial challenge with intraperitoneal inoculation of mucin (Ward 1943). Asheshov et al. (1937) inoculated mice intraperitoneally with a mixture of 109 phage particles and 108 virulent S. Typhi; although the phage and bacteria were allowed to mix in vitro for 10 min prior to inoculation. Two phages in the cocktail were known to attach to the Vi antigen. The mortality rate in control and phage-treated animals was 23/30 and 0/30 respectively. Intravenous inoculation was also effective (mortality rate of 3/30) and, in this case, simultaneous inoculation of bacteria and phage was required since inoculation of phage after 2 or 4 h was less effective (11/30) or ineffective (23/30) respectively.

Other infections involving an intracellular phase are also unlikely to be amenable to phage therapy, once infection has become established. These include plague (Compton 1928), tuberculosis, and brucellosis. Necrotic material may also be present in or around the bacteria, which would reduce the number of productive phage infections, and tissue penetration. The ability of free bacteriophages to penetrate infected macrophages is questionable.

In experimental infections such as this, overwhelming numbers of phages must be used to kill the bacteria in the early stages of infection. This also emphasizes the importance of establishing apposite infection models. The necessity of using large numbers of phages was indicated by Berchieri et al. (1991), in the experimental infection of newly hatched chicks, inoculated orally with different S. Typhimurium strains. Oral inoculation of the birds with high numbers of phages reduced mortality considerably (from 53% to 16% with one strain of Salmonella, F98; and from 60% to 3% with a second strain, 1116), providing that a high multiplicity of infection was used (greater than 10) and that the phages were inoculated within minutes of the Salmonella challenge. In this infection model, the birds die from a typhoid-like infection. This suggests that, in these experiments, the phages were either active in the crop, since passage through the low pH of the gizzard would have inactivated them, or that the bacteria were being killed by nonspecific lysis (abortive infection). There was also considerable variability between groups of birds treated with different phage. Nine phages were tested in total, and mortality ranged from 56% (slightly higher than the control group, 53%) to 16%, p < 0.01. The phages used in this study were not fully characterized, so it is difficult to explain the reason for this discrepancy.

Intestinal E. coli Infections

Smith and Huggins (1983, 1987) explored the use of phages to control enterotoxigenic E. coli in livestock. They were unable to find lytic phages which attached to the K88 and K99 fimbrial antigens of bovine and porcine enterotoxigenic E. coli. Instead, they used phages which were thought to attach to surface LPS such that most mutants that arose would be rough and therefore less virulent, as proved to be the case.

Most of the work was performed with a bovine K99+ strain using two phages administered simultaneously; both of which were virulent, and one of which was able to infect mutants which were resistant to the other phage. Calves (n = 13) infected with 109 E. coli when less than 24 h old all succumbed to diarrhea and subsequently died. None of the nine calves treated with 1011 phage particles 1 h after bacterial challenge became ill. When another group of 13 calves was treated with phage, this time at the onset of diarrhea, only two died. The phages did not completely eliminate the pathogens from the gut but reduced the bacterial numbers to a level which would not produce disease. This has been observed with other experimental work by this and other groups, suggesting that final elimination of the bacteria relied on the host immune response. Similarly, effective treatments were used by this group with pigs and lambs, using strains of E. coli pathogenic for each animal type and isolating virulent phages which infected them (Smith and Huggins 1983).

Other groups have followed this work with pigs showing beneficial effects from either prophylactic administration (with improvements arising from coadministration of florfenicol and an antacid) or therapeutic administration, scoring for clinical and physiological scores and bacterial counts (Jamalludeen et al. 2009). In-feed administration of phage was also found to be an effective prophylactic against experimental challenge (Cha et al. 2012); and in older, weaned animals there were also improvements in clinical scores and weight gain (Lee et al. 2016).

Work with E. coli in cattle has largely centered on O157:H7 strains because of the zoonotic potential of this serotype. Generally, results have been mixed. Rivas et al. (2010) evaluated the ability of two phages to control E. coli O157 in an ex vivo rumen model and cattle of different ages. Both phages were able to significantly reduce pathogen numbers in the ex vivo model, but there was no statistically significant difference in E. coli O157 shedding between phage-treated and control cattle. Rozema et al. (2009) performed a similar trial with experimentally inoculated steers, which were treated orally and/or rectally with a cocktail of four phage which infected E. coli O157. This group also found that there was no significant difference in the shedding of E. coli O157 from control and phage-treated animals. Similar results were obtained in steers by Stanford et al. (2010) who delivered a cocktail of four phages by top-dressing feed or orally delivered in gelatin capsules. The phage appeared to have no effect on bacterial numbers shed but may have reduced the duration of shedding.

In contrast, Sheng et al. (2006) applied a mixture of two virulent phages (25 ml of 1010 PFU/ml preparation) rectally 7 days after rectal administration of four E. coli strains (1010 CFU) to six-month-old Holstein steers, and found significant reduction in shedding (1.3 to 1.5 log10 CFU per swab) compared with the control. These authors also assessed the phages in experimental O157 infection of sheep but found no effect whereas Callaway et al. (2008) found significant reductions 24 h after oral phage administration.

Intestinal Salmonella Infections

The effect of lytic phages on intestinal colonization of chickens by Salmonella Typhimurium has been studied. Berchieri et al. (1991) was unable to demonstrate a significant reduction in the intestinal carriage, or duration of fecal excretion, of S. Typhimurium F98 in 1–2 day-old chickens following oral inoculation with phage ɸAB2; a phage which demonstrated lytic replication on liquid cultures and lawns of this virulent Salmonella strain. The phage was excreted by most birds, but phage numbers fell away much more rapidly than did those of the Salmonella and disappeared when intestinal Salmonella counts fell below 106 CFU/g contents. No phage-neutralizing antibodies were found in the chickens. Several lytic phages administered orally (1011 PFU) to 5-week-old chickens for a few days without antacid, after they were infected orally with S. Typhimurium, had no effect on Salmonella numbers in the ceca (Barrow, Berchieri, and Lovell, unpublished results). In contrast, this group has also shown that virulent phages administered orally with antacid were able to reduce intestinal counts of S. Enteritidis by more than 4 log10 CFU/g and S. Typhimurium by more than 2 log10 CFU/g (Atterbury et al. 2007). However, in this study, resistant mutants developed in vivo following therapy.

Other groups have also reported significant reductions in colonization by Salmonella after administration of phage cocktails. Wong et al. (2014) reduced Salmonella Typhimurium colonization and shedding by intra-cloacal administration of high titre (1012 PFU) phage. Kim et al. (2015a) also reported reductions in colonization by a number of Salmonella serotypes which were either host-specific (Gallinarum, Pullorum) or non-host-specific (Typhimurium, Enteritidis and Derby), in this case detected by a Salmonella-specific PCR signal. Gonçalves et al. (2014) and Bardina et al. (2012) also found a positive effect on intestinal counts and colonization with S. Enteritidis but this required contemporaneous delivery of the phage and bacteria. Bardina et al. (2012) recommended frequent inoculations of phage, ideally just before infection.

Korean groups have centered on the use of phages against experimental fowl typhoid (S. Gallinarum). They have found positive effects on mortality and on tissue counts in contact infections, thus demonstrating that bacterial pathogens, which multiply intracellularly, can be susceptible to phages, but probably only at the initial intestinal infection stage (Hong et al. 2013; Lim et al. 2011). Phages have also been sprayed onto incubating eggs, which if also challenged experimentally, had the effect of reducing clinical disease in hatched chickens (Henriques et al. 2013).

Alternative approaches of phage delivery are also being investigated including the use of phage tail-spike protein, which was demonstrated to reduce chicken colonization by S. Typhimurium (Waseh et al. 2010). Phages with specific attachments sites are also being explored including TolC, a highly conserved protein which forms part of a multidrug efflux pump possessed by Salmonella strains (Ricci and Piddock 2010); and the use of sex pilus-specific phages targeting antibiotic resistant bacteria (Ojala et al. 2016).

In addition to their application in poultry, a small number of experimental trials have been performed on the effect of phage cocktails against Salmonella in pigs, generally against S. Typhimurium. Wall et al. (2010) simultaneously administered 5 × 109 PFU of a phage which had been microencapsulated in alginate beads, and 5 × 108 CFU Salmonella Typhimurium to 3–4-week-old pigs by oral gavage. They reported a 2–3 log10 CFU reduction of bacterial counts in the ileum, cecum, and tonsils. In a further trial, market-weight pigs were first orally inoculated with 5 × 109 CFU S. Typhimurium and then treated with 1010 PFU of a microencapsulated phage suspension 48 h later and comingling with Salmonella-infected pigs (seeders). The phage-treated pigs were readministered phage every 2 h for 6 h (three doses in total per pig) before euthanizing all of the animals and performing bacterial counts. Salmonella counts in the ceca of phage-treated pigs were reduced by 95% compared with untreated controls. Callaway et al. (2011) reported significant reductions (>1.4 log10 CFU) in cecal colonization with direct administration of the phages 24 and 48 h after Salmonella Typhimurium challenge by oral gavage. Albino et al. (2014) recorded a reduction in the number of animals testing positive for Salmonella after phage treatment (from 28/30 to 17/30 animals). Similarly, Saez et al. (2011) reported reductions in the number of chickens shedding Salmonella from 85.7% to 42.9% 4 h after infeed phage administration. Oral administration of the phage by gavage had no effect.

Intestinal Campylobacter Infections

Campylobacteriosis is the most frequently reported bacterial foodborne disease in the developed world and is commonly associated with the consumption of undercooked chicken. Atterbury et al. (2005) found an inverse correlation between the presence of lytic bacteriophages and Campylobacter jejuni counts in the gut of broiler chickens, and several groups have since demonstrated that bacteriophages can be used to reduce the intestinal colonization of chickens by this bacterium. Wagenaar et al. (2005) designed a series of experiments, whereby chicks (~1-week-old) were administered bacteriophages (~1010 PFU) by oral gavage at different times before, during, or after experimental colonization with C. jejuni. A maximum reduction in Campylobacter numbers of 3 log10 CFU/g cecal contents was recorded in phage-treated animals, compared with the control group. The level of Campylobacter reduction in all phage-treated animals, however, tended to converge at 1 log10 CFU by the end of the trial (42 days). Reductions of up to 5 log10 CFU/g cecal content following bacteriophage treatment were reported by Loc Carrillo et al. (2005). However, as with the study by Wagenaar et al. (2005), these reductions were not sustained beyond 2–3 days. Other studies by Carvalho et al. (2010) and El-Shibiny et al. (2009) reported reductions in intestinal colonization by campylobacters of 1–2 log10 CFU following phage treatment.

Perhaps unsurprisingly, some of the above studies found an increase in the percentage of phage-resistant Campylobacter colonies recovered from phage-treated birds, from approximately 6% before phage treatment to 13% after treatment (Carvalho et al. 2010). Both Carvalho et al. (2010) and Loc-Carrillo et al. (2005) found that 54–97% of phage-resistant campylobacters reverted to a phage-sensitive phenotype if they were used to colonize chickens in the absence of phage-selective pressure. This suggests that at least some forms of phage resistance may negatively impact Campylobacter’s ability to colonize intestinal niches in chickens. This interpretation is supported by the results of a study by Scott et al. (2007), which found that phage-resistant campylobacters could outcompete phage-sensitive campylobacters in the presence of bacteriophage predation, but not in the absence of phage. Notwithstanding the problem of phage resistance, if Campylobacter numbers on chicken carcasses were reduced by 2 log10 CFU, then this could be expected to reduce the incidence of campylobacteriosis in humans by 30-fold (Rosenquist et al. (2003). For a more detailed evaluation of the use of phage to control Campylobacter in chickens, see Janez and Loc-Carrillo (2013).

Other Systemic Infections

Dubos et al. (1943) showed that intraperitoneal inoculation of mice, with a crude preparation of phage which infected Shigella dysenteriae, was protective against intracerebral inoculation of the bacterium (mortality was 8/8 for control mice vs 2/8 for phage-treated). There was evidence of phage multiplication in the brain, but protection required early establishment of high phage numbers in circulation. Protection against S. dysenteriae in the highly susceptible chick embryo was also obtained by application of virulent phages (Rakieten and Rakieten 1943).

Biswas et al. (2002) showed that virulent phages administered intraperitoneally protected against infection by Enterococcus faecium administered by the same route. The protective effect was observed even when phage inoculation was delayed until the animals were clinically moribund; where 50% of the mice could be rescued. E. faecium is of considerable clinical significance since excessive use of vancomycin in human medicine, and of the related antibiotic avoparcin as a growth promoter in animal husbandry, has led to an increased risk of high-level resistance to vancomycin in this organism (Bager et al. 1997).

Cultivated fish are also subject to septicemic diseases caused by several bacterial pathogens, and bacteriophages have been used experimentally to treat them (Nakai and Park 2002). Enterococcosis is an economically important disease in cultured Japanese Yellowtail (Seriola quinqueradiata) caused by Lactococcus garviae (formerly Enterococcus seriolicida). Intraperitoneal inoculation of 109 bacteria in yellowtail fish resulted in 55% mortality whereas i.p. inoculation of 107.5 phage 1 h later prolonged the survival of the fish and reduced overall mortality to 10% (Nakai et al. 1999). In a second set of experiments, phages were given to the fish in the form of impregnated feed (containing 108 PFU/g) and were infected with the bacteria 1 h later by anal intubation. Mortality in the control animals was 65%, compared with 20% for the phage-treated animals.

Pseudomonas plecoglossicida produces bacterial hemorrhagic ascites in fish, including the cultivated Ayu (Plecoglossuis altivelis). A study by Nakai and Park (2002) incorporated two phages, originally isolated from diseased fish and pond water, into feed. This feed was administered to the fish, along with inoculation of the bacteria resulted in reduced mortality from 65% to 23% in phage-treated fish. Phage administered 1 or 24 h after the bacteria resulted in a reduction of mortality from 78% to 0% and from 80% to 13%, respectively. Phage-resistant bacterial mutants were recovered, but found to be of reduced virulence. Reduced numbers of bacteria were recovered from the kidneys and water as a result of the different phage treatments. Phages have been found to be effective against a number of other bacterial diseases of fish including Flavobacterium psychrophilum (Castillo et al. 2012) and Aeromonas salmonicida (Kim et al. 2015b) in salmonids, Pseudomonas aeruginosa in catfish (Khairnar et al. 2013), and Vibrio anguillarum in fish larvae (Silva et al. 2014a).

Other Infections

Gabisonia (2001) reported the successful use of intramammary administration of a pool of phage which infected Staphylococcus aureus and Streptococcus pyogenes cured 28/32 cases of bovine mastitis in 7 days; the remaining four cases being cured in another 4 days. However, no details were given of the bacteriology or of control animals. Bovine milk may contain agglutinins, which reduce phage lytic efficacy, and milk proteins, which adhere to the phage particles reducing attachment (Gill et al. 2006a). It remains to be seen whether this is a viable approach to treating mastitis but this group has recorded some improvement in infection after treatment of staphylococcal mastitis although it was not statistically significant (Gill et al. 2006b).

Miller et al. (2010) reported on the use of a cocktail of virulent phages against Clostridium perfringens strains and experimental necrotic enteritis. In 28-day-old birds administered C. perfringens and the phage cocktail by oral gavage, mortality was reduced by 92% and in older 42-day-old birds there were significant gains in both weight and feed conversion.

Use in the Food Production Chain

One veterinary use of phages is in reduction of foodborne pathogens (see chapters “Environmental Hygiene and Bacteriophages” and “Food Safety and Bacteriophages”). Phage therapy is unlikely to be effective in eliminating zoonotic bacterial pathogens from food animals. However, it may be effective in reducing carcass contamination after slaughter, since a chain of production/processing is established in which pathogen-infected animals enter the abattoir/slaughter house, moving in one direction and do not return from a clean to a dirty area. Such treatments have not found a pathway for regulatory approval in the European Union, despite an extensive review of the topic which reported that there were no objections (on safety grounds) to pursuing the use of phage in foods (EFSA 2009). The situation is different in the United States, where several phage products have been approved for the reduction of contamination on live animals, or the direction application of phage to meat and poultry (Goodridge and Bisha 2011; Sarhan and Azzazy 2015). For a list of such approved products, see Table 1.
Table 1

Phage preparations and respective regulatory clearances

Company

Phage product

Target organism(s)

Regulatory

Certifications

FIN TEC GmbH (Hamm, Germany)

Secure Shield E1

E. coli

FDA, GRN 724

 

Intralytix, Inc. (Baltimore, MD, USA)

Ecolicide® (EcolicidePX™)

E. coli O157:H7

USDA, FSIS Directive 7120.1

 

EcoShield™

E. coli O157:H7

FDA, FCN 1018; Israel Ministry of Health; Health Canada

Kosher; Halal

ListShield™

L. monocytogenes

FDA, 21 CFR 172.785; FDA, GRN 528; EPA Reg. No. 74234-1; Israel Ministry of Health; Health Canada

Kosher; Halal; OMRI

SalmoFresh™

Salmonella spp.

FDA, GRN 435; USDA, FSIS Directive 7120.1; Israel Ministry of Health; Health Canada

Kosher; Halal; OMRI

ShigaShield™ (ShigActive™)

Shigella spp.

FDA, GRN 672

 

Micreos Food Safety (Wageningen, Netherlands)

PhageGuard Listex™

L. monocytogenes

FDA, GRN 198/218; FSANZ; EFSA; Swiss BAG; Israel Ministry of Health; Health Canada

Kosher; Halal; OMRI; SKAL

PhageGuard S™

Salmonella spp.

FDA, GRN 468; FSANZ; Swiss BAG; Israel Ministry of Health; Health Canada

Kosher; Halal; OMRI; SKAL

PhageGuard E™

E. coli O157:H7

FDA, GRN 757

 

Passport Food Safety Solutions (West Des Moines, IA, USA)

Finalyse®

E. coli O157:H7

USDA, FSIS Directive 7120.1

 

Phagelux (Shanghai, China)

AgriPhage™

Xanthomonas campestris pv. vesicatoria, Pseudomonas syringae pv. tomato

EPA Reg. No. 67986-1

 

SalmoPro®

Salmonella spp.

FDA, GRN 603

 

SalmoPro®

Salmonella spp.

FDA, GRN 752

 
 

E. coli spp.

FDA, GRN 827 pending as of February 25, 2019

 

Several studies have investigated the use of phages to reduce pathogens on experimentally contaminated meat and skin. One study showed reduction in viable numbers of S. Enteritidis, using artificially contaminated carcass skins (Goode et al. 2003). It was possible to eliminate detectable Salmonella organisms from skin samples by spraying the surface with high titre suspensions of phage. Other related serotypes in this study were also susceptible to phage attachment and penetration because of a common O-antigen, although the phage could not replicate productively on these strains, possibly because of differences in restriction modification among Salmonella strains. Similar results have also been obtained using this approach with C. jejuni on chicken skin (Atterbury et al. 2003) and S. Typhimurium on pig skin (Hooton et al. 2011). These studies demonstrated a reduction of the targeted pathogens to below detectable limits. This approach has also been used to reduce Salmonella contamination of other foods such as smoked salmon (Galarce et al. 2014). Both Goode et al. (2003) and Hooton et al. (2011) attribute the reduction in Salmonella contamination to nonproductive infection (“lysis from without” and “passive therapy” respectively).

Lysis from without was a term originally used to describe observations in the laboratory setting of bacterial lysis induced by high-multiplicity virion adsorption, lysis that occurs without production of phage progeny. The term “abortive infection” may be used more generally to describe unproductive phage infections that kill bacteria (Abedon 2011). Speculation as to the impact of lysis from without in phage therapy is common. This speculation potentially is a consequence of similarities between lysis from without and “passive treatment,” contrasting with “active treatment” where bacteriophage replication is a key part of the approach. Though both passive treatment and lysis from without involve high multiplicities of phage adsorption, the main difference is that passive therapy allows for internal effects along with phage replication, while lysis from without as usually defined is simply a cell-envelope effect resulting from repeated enzymatic “holing” of the bacterial cell wall. As historically defined, lysis from without involves extremely high ratios of phages adsorbed to the bacterial target, typically 100:1 or more. Since such ratios are very unlikely to be achieved consistently in vivo, the actual significance of lysis from without other than as a laboratory observation is questionable.

A number of studies have chosen components of bacteriophages to treat infections, rather than intact viruses, which is covered elsewhere in this work (see chapter “Enzybiotics: Endolysins and Bacteriocins”).

The use of bacteriophages either for directly killing bacteria through the lytic infection cycle or following abortive infection have now been applied to a number of food stuffs post-slaughter, either to reduce spoilage (Li et al. 2014; Pérez Pulido et al. 2015) or eliminate other foodborne pathogens, particularly Listeria, on fish (Soni and Nannapaneni 2010); cheese (Silva et al. 2014b), in this case using a commercially available phage product, which has been characterized in detail (Carlton et al. 2005); milk (Rodríguez-Rubio et al. 2015); fruit (Oliveira et al. 2014); cooked meat (Chibeu et al. 2013); and in the formation of biofilms (Ganegama Arachchi et al. 2013; Khalifa et al. 2015). Further information on the use of phage to counter food-spoilage can be found elsewhere in this work (see chapter “Food Safety and Bacteriophages”).

Applications, Potential, and Problems

From the experimental and fieldwork on infectious disease described here, it would seem that phage therapy and prophylaxis can be very successful, and in some cases can be more effective than antibiotics. However, greater efficacy is more likely in an environment which mimics those conditions under which phages multiply optimally in vitro. In terms of disseminating phage around the body, liquid tissues such as blood or cerebrospinal fluid would be favored or environments associated with skin and intestinal mucosae, where phages can be transmitted between bacteria by close or direct contact, as they are on a bacterial lawn on the surface of an agar plate (see Detection of Bacteriophages: Plaques and Plaque Assay). However, unlike in microbiological media, the mammalian immune system is able to remove phage particles from the circulatory system, so care needs to be taken to select phage which are both efficacious and persistent in this environment (Merril et al. 1996). Such phages are likely to be successful in controlling bacterial growth in vivo in septicemia and meningitis, and in gastrointestinal infections where the bacteria colonize the intestinal mucosae, as occurs with ETEC and cholera (Yen et al. 2017; Bhandare et al. 2019) in humans and in bacterial colonization of the skin and mucous membranes.

It seems unlikely that phage therapy will be successful if access to target bacteria is limited, e.g., intracellular bacteria such as mycobacteria, Brucella, or Yersinia (including plague). Animal typhoid infections caused by various Salmonella enterica serotypes may also be included in this category, although prevention of infection at the mucous membranes before they become intracellular is a possibility, and d’Herelle’s original work suggests that this should be reexplored (d’Herelle 1926). Phage therapy is also unlikely to be successful where the target bacteria are surrounded by large amounts of inert material, as occurs with cellular debris in areas of inflammation, or in the large bowel where the target bacteria (such as enterococci, for example) may be outnumbered by other bacteria in ratios of more than 1000 to 1,000,000 to 1.

An added complication is resistant subpopulations of bacteria. Phages which are active against bacterial strains, which have the correct attachment determinant, may nevertheless be resistant because of DNA restriction/CRISPR/Cas activity (see chapter “Bacteria-Phage Antagonistic Coevolution and Implications for Phage Therapy”). Phages can be adapted to new strains by passage. However, it does mean that, in many cases, pathogens will have to be screened against a bank of phages to find those which are virulent for the targeted strains of pathogen. Variations in DNA restriction explain at least some of the variability in in vitro lysis and in vivo effectiveness found by Smith and Huggins (1982). Consequently, if a pathogen is largely clonal during an epidemic, then this problem can be avoided. Clonality and the problem of DNA restriction can also be addressed in part by administering such a high dose of phage that each target bacterium is attacked by many phages, leading to abortive infection, as likely occurred in the Salmonella studies on chicken and pig skin and the numerous publications on their use against food-spoilage organisms or foodborne pathogens mentioned above. It is unlikely that phages will be a practical option for neonatal enteritis in young animals since the problems of nonclonality are compounded by the fact that a range of pathogens may cause this disease, including viruses and protozoans which will not be susceptible to phage.

Ideally, to maximize the success of phage treatments, the animal patient should be an epidemiological end point, such that cycling of the pathogen and phage in the environment and other hosts does not occur, and opportunities for the development of phage resistance are therefore limited. This is the case with most systemic diseases and also in the case of human infections, with colonization of skin burns involving isolation of the patient and a reduced chance for spread. It can be reduced for some enteric pathogens by collecting the feces of the treated livestock, preventing their release into the environment. In relation to veterinary applications, phage treatments for septicemia could be revisited, especially where outbreaks are caused by a single bacterial strain. The bacterial pathogens that may be amenable to this approach include Pasteurella multocida in poultry where it may be an economic option in turkeys, P. septicaemia in calves and lambs, and Riemerella anatipestifer in ducks and geese.

E. coli produces septicemia and other systemic infections, often following viral infections in poultry. The experimental work suggests that these would be appropriate for phage therapy or prophylaxis, providing the problems of clonality can be addressed. Pathogens where the main site of infection is intracellular, and which rarely lead to bacteremia until the final stages of the disease, are unlikely to be treated successfully. This includes bacteria such as Brucella, Yersinia, host-restricted serotypes of Salmonella, and mycobacteria as indicated above. Prophylactic treatment is also likely to be unsuccessful against such pathogens because of recirculation of the phage and bacteria in the environment, which may lead to resistance development. Some success has been obtained with a mouse model of equine keratitis but it is unclear how far this involves intracellular activity (Furusawa et al. 2016).

Phage therapy is currently being contemplated for use against foodborne Salmonella serotypes and Campylobacter jejuni in poultry. It is unlikely, however, to be able to eliminate totally the bacteria from the gut, which would be the desirable outcome of treatment. In addition, despite the reductions seen in experimental work, in the field excretion of both pathogen and phage in the feces would result in rapid infection of the next flock to populate the house after the treated birds had been taken to slaughter. This cycling would facilitate the development of resistance. However, phages may have a use in reducing transmission of these zoonotic pathogens from animal to humans via the food chain by the treatment of carcasses with phage preparations immediately after slaughter and evisceration (see chapter “Food Safety and Bacteriophages”). This is a situation where the number of target bacteria present on the skin is low and relatively accessible. Spraying with a high titre phage suspension or carcass dipping may significantly reduce the pathogen load in order to achieve a measurable public health benefit. The issue of resistance development is likely to be minimal since the phages would not find their way back to farms in any numbers. A major obstacle to phage treatments for food animals, at least in the European Union, is compatibility of phage treatments with current legal frameworks and definitions, and the acceptability of phage biocontrol to regulators.

Regulatory Approval

From farm-to-fork, there are many steps where bacteriophages might be used to manage bacterial populations, but before being used it is necessary to have the approval of the competent authorities. The usage of phage-derived products, such as phage endolysins against foodborne pathogenic bacteria (Schmelcher et al. 2015) or the usage of phages to reduce food spoilage (Greer 2005) were subject of consideration and review elsewhere. Regulation of the use of bacteriophages in a veterinary context, including that of food production and the food chain, is gradually changing. The current phage-approved products fall in the second category and are deemed to be used in the post-slaughter/harvest and storage (e.g., sprayed directly on food surfaces, packaging material, or other surfaces) to reduce the contamination by the targeted bacteria (Sulakvelidze 2013; Lone et al. 2016). Table 2 provides a list of studies where phages have been applied to foods, including phage which have been approved as commercial products in at least one country.
Table 2

Studies that employ phage preparations designed to be applied directly on food. Sorted by organism and year of publication

Target

Phages

Details

Reference

Bacillus cereus

BCP1-1

Single phage applied in fermented soya bean paste decreased Bacillus cereus counts without impact on Bacillus subtilis which is important during the product fermentation process

Bandara et al. (2012)

Campylobacter jejuni

Φ2

Single phage applied on chicken skin at 4 °C reduced Campylobacter counts by ~1 log

Atterbury et al. (2003)

Campylobacter jejuni

Salmonella spp.

C. jejuni typing page 12673, P22, 29C; Salmonella typing phage 12

Each phage applied to experimentally contaminated chicken skin decreased the recoverable bacterial numbers by up to 2 log when using a MOI of 100 or higher. Using a MOI of 105 and <100 Salmonella CFU/unit area of skin, Salmonella contamination was eliminated completely

Goode et al. (2003)

Campylobacter jejuni

Salmonella spp.

Cj6

P7

Phage Cj6 applied on beef significantly decreased the campylobacter contamination, however for low levels of contamination (~100 CFU/cm2) the bacterial count reduction was not significative

Phage P7 applied on cooked beef at 5 °C decreased Salmonella counts by ~2–3 log, while used on raw beef at 24 °C the reduction was >5.9 log

Each phage was more effective at MOI 10000:1 and ~10,000 CFU/cm2 of bacteria

Bigwood et al. (2008)

Cronobacter sakazakii

ESP 1-3, ESP 732-1

Phage applied on infant formula at 37 °C, 24 °C, and 12 °C reduced the contamination level of Cronobacter sakazakii. The reduction was more effective at 24 °C

Kim et al. (2007)

Cronobacter sakazakii

Five phages

Phage cocktail with five phages applied in experimentally contaminated infant formula inhibited the growth of Cronobacter sakazakii. Each individual phage (108 PFU/ml) eliminated C. skazakii in liquid culture at 106 and 102 CFU/ml

Zuber et al. (2008)

Escherichia coli O157:H7

e11/2, pp01, e4/1c

Phage cocktail with three phages applied on the surface of experimentally contaminated (103 CFU/ml) beef at 37 °C eliminated E. coli O157:H7 from the majority of treated samples

O’Flynn et al. (2004)

Escherichia coli O157:H7

EcoShield™ (formerly ECP-100)

Phage cocktail applied to broccoli, spinach, and tomatoes decreased the level of contamination of E. coli O157:H7 by ~1–3 log or below the limit of detection. When used on ground beef, the reduction was ~1 log

Abuladze et al. (2008)

Escherichia coli O157:H7

EcoShield™ (formerly ECP-100)

Phage cocktail applied to experimentally contaminated lettuce and cantaloupe reduced significantly the counts of E. coli O157:H7 by up to 1.9 log and 2.5 log, respectively

Sharma et al. (2009)

Escherichia coli O157:H7

Cocktail BEC8

Phage cocktail applied on the surface of leafy green vegetables at 37, 23, 8, and 4 °C reduced the contamination with E. coli O157:H7 by ~2–4 log

Viazis et al. (2011)

Escherichia coli O157:H7

EcoShield™ (formerly ECP-100)

Phage cocktail applied on the surface of experimentally contaminated lettuce and beef reduced the level of contamination with E. coli O157:H7 by ~87% and 94%, respectively. A single treatment did not protect the foods after experimental recontamination with the same bacteria

Carter et al. (2012)

Escherichia coli O157:H7

EcoShield™ (formerly ECP-100)

Phage cocktail applied on the surface of leafy greens at 4 and 10 °C under modified ambient and atmosphere reduced by >2 log the level of E. coli O157:H7

Boyacioglu et al. (2013)

Escherichia coli

FAHEc1

Phage applied on raw and cooked beef at 37, 24, and 5 °C reduced the counts of E. coli

Hudson et al. (2013)

Escherichia coli O157:H7

EcoShield™ (formerly ECP-100)

Phage cocktail applied to lettuce via dipping or spraying. The dipping process consists of a 2 min immersion in phage suspension, and showed a reduction of ~0.7 log in Escherichia coli O157:H7 counts

The spray process showed a larger initial reduction (~0.8–1.3 logs) in E. coli O157:H7 counts

Ferguson et al. (2013)

Escherichia coli

EC6, EC9, EC11

Phage cocktail with three phages applied to UHT milk showed to eliminate two E. coli strains at 5–9 °C and 25 °C. With another E. coli strain, counts were reduced initially; however, regrowth occurred in the raw milk after 144 or 9 h at 5–9 °C or 25 °C, respectively

McLean et al. (2013)

Escherichia coli, Salmonella, Shigella

EcoShield™ (formerly ECP-100), SalmoFresh™, ShigActive™

Phage cocktails used in combination with produce wash reduced targeted pathogenic bacteria from broccoli, cantaloupe, and strawberries

The phage treatment was as effective or more than chlorine washing at reducing targeted bacterial counts

Magnone et al. (2013)

Listeria monocytogenes

ListShield™ (formerly LMP-102)

Phage cocktail applied on the surface of melon and apple slices reduced the counts of Listeria by ~2 log and ~0.4 log, respectively. The use of Nisin revealed a synergistic effect on contamination reductions of ~5.7 log and ~2.3 log, respectively

Leverentz et al. (2003)

Listeria monocytogenes

ListShield™ (formerly LMP-102)

Single phage applied preventively on the surface of honeydew melon tissue and after 1, 0.5, or 0 h experimentally contaminated with Listeria effectively reduced bacterial counts by ~5–7 log after 7 days. The phage efficacy was shown to be concentration dependent

Leverentz et al. (2004)

Listeria monocytogenes

PhageGuard Listex™ (formerly Listex™; P100)

Phage cocktail applied on the surface of ripened red-smear cheese reduced the counts of Listeria by at least 3.5 log. Isolated colonies of Listeria after the phage treatment were not resistant to the phage

Carlton et al. (2005)

Listeria monocytogenes

A511, PhageGuard Listex™ (formerly Listex™; P100)

Single phage applied to experimentally contaminated chocolate milk and mozzarella cheese brine at 6 °C eradicated Listeria.

Phage cocktail applied to sliced cabbage, iceberg lettuce, mixed seafood, smoked salmon, sliced turkey meat, and hot dogs reduced Listeria counts up to 5 log

Guenther et al. (2009)

Listeria monocytogenes

PhageGuard Listex™ (formerly Listex™; P100)

Single phage applied to the surface of red salmon (~108 PFU/g) at 4 or 22 °C reduced the Listeria counts by ~1.8–3.5 log

Soni and Nannapaneni (2010)

Listeria monocytogenes

ListShield™ (formerly LMP-102) PhageGuard Listex™ (formerly Listex™; P100)

Single phage application on the surface of raw catfish fillets at 22, 10, and 4 °C reduced the level of Listeria contamination by approximately 1.6–2.3, 1.7–2.1, and 1.8–3.5 log CFU/g, respectively

Soni et al. (2010)

Listeria monocytogenes

A511

Single phage applied to soft cheese reduced Listeria contamination by 2 log. Subsequent phage administrations did not decrease the natural microbial community nor decreased Listeria any further

Guenther and Loessner (2011)

Listeria monocytogenes

FWLLm1

Single phage applied to surface of experimentally contaminated chicken at 30 or 4 °C stored in vacuum packages decreased the counts of Listeria by 1–2 log. Listeria regrowth was observed only in samples stored at 30 °C

Bigot et al. (2011)

Listeria monocytogenes

PhageGuard Listex™ (formerly Listex™; P100)

Single phage applied to experimentally contaminated queso fresco decreased the counts of Listeria by ~3 log but contamination regrowth was observed. Simultaneous administration of antimicrobials potassium lactate and sodium diacetate with the phage had similar effect on Listeria reduction and prevented bacteria regrowth. The simultaneous administration of lauric arginate and the phage had reduced effect on count reduction and did not prevented Listeria regrowth

Soni et al. (2012)

Listeria monocytogenes

PhageGuard Listex™ (formerly Listex™; P100)

Single phage applied to ready-to-eat beef and turkey at 10 or 4 °C decreased the counts of Listeria; however, subsequent regrowth was observed at both temperatures. The phage treatment was more effective than the usage of potassium lactate or potassium lactate-sodium diacetate mixture. The usage of the mentioned antimicrobials combined with the phage application effectively reduced the Listeria counts and prevented bacterial regrowth at both temperatures tested

Chibeu et al. (2013)

Listeria monocytogenes

PhageGuard Listex™ (formerly Listex™; P100)

Single phage applied to experimentally contaminated melon and pear at 10 °C reduced the counts of Listeria by ~ 1.5 log after 2 days. The phage treatment was not effective on apple slice. The phage applied to melon juice reduced the counts of Listeria by ~4 log, but in pear juice the reduction was only ~2.5 log

Oliveira et al. (2014)

Listeria monocytogenes

PhageGuard Listex™ (formerly Listex™; P100)

Single phage applied to soft cheese at 10 °C reduced the counts of Listeria by ~2–3 log after 30 min and ~0.8–1 after 7 days

Silva et al. (2014b)

Listeria monocytogenes

ListShield™ (formerly LMP-102)

Phage cocktail applied to experimentally contaminated lettuce and cheese reduced the Listeria counts after 5 min by 1.1 and 0.7 log, respectively. When applied simultaneous with antioxidant solution on the surface of apple slices, after 24 h, the Listeria counts were reduced 1.1 log

The phage cocktail showed to be effective reducing to undetectable levels of Listeria contamination on frozen entrees and environmental contamination at a smoked salmon factory

Perera et al. (2015)

Listeria monocytogenes

PhageGuard Listex™ (formerly Listex™; P100)

Phage cocktail applied to the surface of experimentally contaminated sliced pork ham reduced Listeria below the detectable limits after 72 h. The phage treatment was more effective reducing Listeria counts than the usage of nisin, sodium lactate individually, or in combination

Figueiredo and Almeida (2017)

Mycobacterium smegmatis

Six phages

Phage cocktail with six phages or each phage individually applied to milk reduced M. smegmatis counts bellow the limit of detection. When stored at 37 °C, after 96 h bacterial regrowth was observed when using single phage treatment; however, no regrowth occurred when using the phage cocktail

Endersen et al. (2013)

Salmonella spp.

SJ2

Single phage applied to milk before cheese production reduced the counts of Salmonella in cheese by 1–2 log, when compared to control milk without phage treatment, the final cheese product had Salmonella counts ~1 log higher

Modi et al. (2001)

Salmonella spp.

SCPLX-1

Phage cocktail with four phages applied to melon slices at 20 or 10 and 5 °C reduced the Salmonella counts by ~2.5 and ~3.5 log, respectively. The phage cocktail had no effect on Salmonella contamination reduction when used on apple slices

Leverentz et al. (2001)

Salmonella spp.

Felix-O1

Single phage applied to experimentally contaminated chicken frankfurters significantly reduced the counts of Salmonella Typhimurium DT104 by 2.1 log

Whichard et al. (2003)

Salmonella spp.

PHL4

Single phage applied to naturally contaminated turkey carcasses or experimentally contaminated broiler carcasses reduced the Salmonella contamination by 60% and 100%, respectively

Higgins et al. (2005)

Salmonella spp.

Six phages

Phage cocktail with six phages applied to experimentally contaminated mung bean sprouts reduced the level of contamination by ~3 log. The simultaneous application of phage and antagonistic bacteria Enterobacter asburiae reduced the Salmonella counts by ~6 log

Ye et al. (2010)

Salmonella spp.

FO1-E2

Single phage applied to chocolate milk and mixed seafood at 8 °C reduced the Salmonella counts after 24 h below the limit of detection. No regrowth was detected

Single phage applied to sliced turkey breast, chocolate milk, and hotdogs at 15 °C reduced Salmonella counts within 24–48 h below the limit of detection. No regrowth was detected within 5 days

Single phage applied to egg yolk and mixed seafood reduced Salmonella counts by ~0.5–2 and ~1–3 log, respectively. No regrowth occurred on mixed seafood; however, in egg yolk regrowth occurred in just 2 days to phage-untreated control

Guenther et al. (2012)

Salmonella spp.

UAB_Phi 20, UAB_Phi78, UAB_Phi87

Phage cocktail applied on fresh eggs shells, lettuce reduced the Salmonella counts by~1 and ~2–4 log, respectively. Phage cocktail suspension used to dip chicken breasts in and afterwards stored at 4 °C, reduced the Salmonella counts by 1–2 log with no regrowth within 7 days

Phage cocktail applied on pig skin reduced Salmonella counts after 6 h at 33 °C by 2–4 log

Spricigo et al. (2013)

Salmonella spp.

wksl3

Single phage applied to chicken skin reduced the counts of Salmonella by ~3 log with no change in the bacterial levels within 7 days at 8 °C

Kang et al. (2013)

Salmonella spp.

Five phages

Phage cocktail with five phages applied on chicken breast reduced Salmonella counts by ~1 log

Hungaro et al. (2013)

Salmonella spp.

P22

Single temperate phage reduced Salmonella counts when administered followed by storage at 4 °C on chicken (~0.5–2 log), whole or skimmed milk (below the detection limit), apple juice (~2 log), liquid egg (~2 log), and energy drink (~2 log)

Zinno et al. (2014)

Salmonella spp.

SalmoFresh™

Phage cocktail sprayed on chicken skin, previously washed with chlorine or peracetic acid, reduced significantly Salmonella counts by ~2.5 log. The usage of only the phage cocktail, chlorine, or low levels of peracetic acid resulted in Salmonella counts reduction of 0.5–2.5 log

Sukumaran et al. (2015)

Salmonella spp.

SalmoFresh™

Phage cocktail applied by dipping or on the surface of chicken breast fillets followed by 4 °C storage significantly reduced the counts of Salmonella by ~0.9 log. Storage in modified atmosphere package resulted in Salmonella counts reduction of up to 1.2 log

Sukumaran et al. (2016)

Salmonella spp.

SalmoLyse™

Phage cocktail applied to the surface of raw pet food ingredients (chicken, tuna, turkey, cantaloupe, and lettuce) reduced the level of Salmonella contamination by ~0.4–1.1 log

Soffer et al. (2016)

Salmonella spp.

SJ2

Single phage applied to experimentally contained ground pork and eggs reduced the number of Salmonella colonies isolated after phage treatment at room temperature and 4 °C. Significantly more phage-resistant colonies were isolated after phage treatment on egg compared to ground pork

Hong et al. (2016)

Salmonella spp.

PhageGuard S™ (formerly Salmonelex™)

Phage cocktail applied to experimentally contaminated chicken thighs and legs achieved a large reduction in Salmonella counts, especially on Salmonella serovars isolated from other sources and not from ground chicken

Grant et al. (2017)

Salmonella spp.

PhageGuard S™ (formerly Salmonelex™)

Single phage applied to ground beef trim significantly reduced Salmonella counts by ~1 log, comparable to the result of using irradiation alone. Using irradiation followed by phage treatment resulted in a ~2 log reduction of Salmonella counts

Yeh et al. (2018)

Shigella spp.

SD-11, SF-A2, SS-92

Phage cocktail or each phage individually applied to spiced chicken and stored at 4 °C reduced Salmonella counts by ~1–4 log

Zhang et al. (2013)

Shigella sonnei

ShigaShield™

Phage cocktail with five phages applied to ready-to-eat foods (melon, corned beef, smoked salmon, precooked chicken) reduced the recovery of Shigella ~1–1.4 log when compared to control

Soffer et al. (2017)

Staphylococcus aureus

Φ 35, Φ 88

Phage cocktail with two phages applied to whole milk and stored at 37 °C reduced Staphylococcus aureus below detectable limits. Phage treatment eliminated S. aureus from the acid curd after storage at 25 °C after 4 h, and from the renneted curd at 30 °C after 1 h

Garcia et al. (2007)

Staphylococcus aureus

vB_SauS-phi-IPLA35, vB_SauS-phi-SauS-IPLA88

Phage cocktail with two phages applied to experimentally contaminated milk reduced the counts of S. aureus on subsequent cheese products. Starter strains were not affected by the presence of phages in the cheese making processes and cheeses maintained their expected physical and chemical properties

Bueno et al. (2012)

It was only in 2006 that the first bacteriophage preparation was approved to be used directly in the food supply. Issued by the US agency Food and Drugs Administration, the phage cocktail trademarked as ListShield™ obtained clearance to be used as a food additive for “their intended applications,” which is to reduce Listeria monocytogenes contamination in food. That same year, another phage cocktail against Listeria, Listex™ (currently PhageGuard Listex™) obtained for FDA a Generally Regarded as Safe (GRAS) designation, which is a no-objection letter. Recently, the FDA have considered more phage cocktails preparations as GRAS under the intended conditions of use (e.g., SalmoPro® 26 Jul 2015, ShigaShield 27 Mar 2017, Secure Shield E1 10 Apr 2018, PhageGuard E™ 8 Aug 2018).

Currently, it seems that the legal environment of approval of phage products to food usage (post-harvest) follows the main path to obtain a GRAS clearance notice, at least in the United States. This seems to be the case because the current phage products are not genetically modified, in fact the phages used are all wild-type isolated from and available in nature; consequentially, it is logical that a clearance as regarded as safe seems to be appropriate. As more and more phage preparations obtain a GRAS clearance, other institutions, such as United States Department of Agriculture (USDA), have adapted their guidelines to incorporate the changes. The USDA branch of Food Safety and Inspection Service (FSIS) in the FSIS Directive 7120.1 lists the safe and suitable ingredients used in the production of meat, poultry, and egg products, and it includes now 13 phage preparations. These preparations include, for example, bacteriophage preparation (targeting E. coli O157:H7) to be applied as a spray, mist, rinse, or wash to the hides of live animal (cattle) within lairage, restraining areas, stunning areas, and other stations prior to hide removal; bacteriophage preparations (Salmonella targeted) applied as a spray mist or wash on the feathers of live poultry prior to slaughter; and bacteriophage preparations (against L. monocytogenes) applied as a spray on product surface area of various ready-to-eat meat and poultry products.

Only in 2016, the European Food Safety Authority (EFSA) have issued the first safety and efficacy report about a phage preparation commercially available (PhageGuard Listex™). As in the United States, regulators have issued approvals for phage preparations; other countries are also following the US lead and issuing approvals for the same products in their own countries (Australia, Canada, New Zealand, and Switzerland) or member states (European Union).

In summary, there seems to be considerable potential for using bacteriophages for the treatment and prevention of animal infections, providing that certain criteria are fulfilled. It would seem appropriate now, for research funding, to consider phage use in veterinary contexts as a means to combat antibiotic resistance in bacteria, which are amenable to phage therapy.

Cross-References

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.School of Veterinary Medicine and ScienceUniversity of NottinghamLeicestershireUK

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