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ARTICLE IN PRESS International Journal of Medical Microbiology 296 (2006) 5–14 www.elsevier.de/ijmm REVIEW Phage therapy: Facts and fiction Mikael Skurnika,!, Eckhard Strauchb a Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, P.O. Box 21, FIN-00014, and Helsinki University Central Hospital Laboratory Diagnostics, Helsinki, Finland b Federal Institute for Risk Assessment, Berlin, Germany Received 15 April 2005; received in revised form 5 September 2005; accepted 12 September 2005 Abstract Recent examples of the use of bacteriophages in controlling bacterial infections are presented, some of which show therapeutic promise. The therapeutic use of bacteriophages, possibly in combination with antibiotics, may be a valuable approach. However, it is also quite clear that the safe and controlled use of phage therapy will require detailed information on the properties and behavior of specific phage–bacterium systems, both in vitro and especially in vivo. In vivo susceptibility of bacterial pathogens to bacteriophages is still largely poorly understood and future research on more phage–bacterium systems has to be undertaken to define the requirements for successful phage treatments. r 2005 Elsevier GmbH. All rights reserved. Keywords: Phage therapy; Yersinia enterocolitica; Escherichia coli Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . Principles of phage biology . . . . . . . . . . . . . Bacteriophage receptors and phage resistance Prerequisites for phage therapy . . . . . . . . . . Phage (pharmaco)kinetics . . . . . . . . . . . . . . Phages may carry harmful genes . . . . . . . . . Phage products . . . . . . . . . . . . . . . . . . . . . Recent in vivo phage studies . . . . . . . . . . . . Phage therapy trials in chickens . . . . . . . . . . Phage therapy in fish aquacultures . . . . . . . . Phages riding inside a Trojan horse . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !Corresponding author. Tel.: +358 9 1912 6464; fax: +358 9 1912 6382. E-mail address: [email protected] (M. Skurnik). 1438-4221/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2005.09.002 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 .6 .7 .7 .7 .8 .8 .9 10 11 11 11 11 ARTICLE IN PRESS 6 M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14 Introduction Principles of phage biology In the April 2003 issue of ‘‘New Scientist’’, James Randerson wrote an article ‘‘Virus cleans up food poisoning bug’’ (Randerson, 2003) where he reported observations presented by Andrew Brabban (Evergreen State College in Washington state) in the XXth Annual Meeting of the Society for General Microbiology in Edinburgh, UK. Brabban had wanted to test the effect of different antibiotics on Escherichia coli O157:H7 in infected sheep. However, the researchers faced an unexpected problem; the bacteria disappeared from the infected sheep very rapidly. It turned out that the sheep carried a bacteriophage specific for E. coli O157:H7 and the phage efficiently eliminated all the inoculated pathogens from the sheep tissues. This is an example of a number of observations made on the antibacterial potency of bacteriophages during the last 90 years after the independent discoveries of bacteriophages by d’Herelle and Thwort. The prospect of phage therapy has stimulated a lot of discussion recently. The increase in interest can be explained in part by the publication of experiments conducted using phage lysins (Loeffler et al., 2001; Nelson et al., 2001; Schuch et al., 2002) and by animal experiments where the bacterial infections are challenged with live bacteriophage particles (Biswas et al., 2002; Broxmeyer et al., 2002; Cerveny et al., 2002; Loeffler et al., 2003; Merril et al., 1996; Sulakvelidze et al., 2001; Westwater et al., 2003). Steve Projan (2004) presented a number of provocative arguments recently challenging the optimism regarding phage therapy. Apparently, Projan dislikes proponents of phage therapy since he puts forward such expressions like ‘‘a cult of phage therapy followers’’, ‘‘the little animal efficacy data there is in the literature can charitably be described as meager’’, ‘‘this silence (on animal efficiency data) speaks volumes’’ and ‘‘anecdotal testimonials of former patients’’. We will not try to cover old literature in this review since there are excellent recent reviews on phage therapy available (Alisky et al., 1998; Anonymous, 1983; Bradbury, 2004; Bull et al., 2002; Cerveny et al., 2002; Dixon, 2004; Duckworth and Gulig, 2002; Inal, 2003; Levin and Bull, 2004; Merril et al., 2003; Nakai and Park, 2002; Payne et al., 2000; Pirisi, 2000; Projan, 2004; Schoolnik et al., 2004; Stone, 2002a, b; Sulakvelidze et al., 2001; Summers, 2001; Thacker, 2003; Thiel, 2004; WeberDabrowska et al., 2002, 2003; Weinberg, 2002). We will focus mainly on recent work carried out to exploit the therapeutic potential of phages and phage products to combat bacterial pathogens. Another interesting aspect of phage application is the use of whole phage particles to deliver vaccines in the form of immunogenic peptides attached to modified phage coat proteins or as delivery vehicles for DNA vaccines, which was recently reviewed by Clark and March (2004). Bacterial viruses (bacteriophages) occupy all those habitats of the world where bacteria thrive. It has been estimated that for each bacterial cell there are ten bacteriophage particles. Recently, the existence of viruses specific for archaebacteria (archeophages) has become evident also. Some phages are highly specific while others are extremely broad in their host range. Bacteriophage taxonomy is based on their shape and size as well as on their nucleic acid. Most bacteriophages have ds DNA, however, some have ss DNA, ds RNA or ss RNA. Upon infection of the bacterial host different phages can have quite different fates. Some phages follow the lytic infection cycle whereby they multiply in the bacterial cell and lyse the bacterial cell at the end of the cycle to release newly formed phage particles. Some phages may use the lysogenic pathway where the phage genome will integrate as part of the host genome, replicate as part of the host genome and stay in a dormant state as a prophage for extended periods of time. If the host bacterium encounters adverse environmental conditions the prophage may become activated and turn on the lytic cycle, at the end of which the newly formed phage particles will lyse the host cell. The following phases can be distinguished in the lytic bacteriophage developmental cycle: 1. Adsorption of the phage on the bacterial cell by binding to a specific receptor. 2. Injection of the nucleic acid into the bacterium. 3. Expression of the phage early genes, synthesis of early proteins, most involved in the shutting down of the host bacterium systems and phage genome replication. 4. Replication of the phage genome. 5. Expression of the phage late proteins involved in the formation of new phage particles and lysis of the host bacterium. 6. Assembly of the phage heads and tails and packaging of the genome. 7. Lysis of the host bacterium and release of the new phage progeny. The ability of the phages to kill the bacterial cells at the end of the infectious cycle is the cornerstone of the idea of using phages as therapeutic agents. However, for a positive outcome of the therapeutic use of bacteriophages all the above listed steps in the phage infectious cycle need to take place. Bacteriophages have coevolved with their bacterial hosts. Already in the few thoroughly studied phage–bacterium systems many examples of intricate molecular mechanisms have been revealed. Therefore, one cannot expect that all the phage/host systems will behave identically under the conditions met ARTICLE IN PRESS M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14 in vivo; on the contrary, it would be surprising to find two identically behaving systems. Bacteriophage receptors and phage resistance One essential step in phage infection is the attachment of the phage onto the bacterium. For this purpose phages can use bacterial capsules, different parts of lipopolysaccharide (LPS), flagella, fimbriae and many other surface proteins as receptors. Bacteriophages may also use enzymes to break down capsule-like materials on the bacterial surface in a drill-like manner to reach the cell wall of the bacterium. A good example of such a phage is PK1A that uses the endosialidase activity of its tail to degrade the polysialic acid capsule of E. coli K1 (Pelkonen et al., 1992). By mutating or losing the phage receptor bacteria become resistant to phages in question. This may not always be bad (Levin and Bull, 2004) since resistance may reduce the fitness of the bacteria or if the receptor used by the phage is a virulence determinant, loss of the receptor would decrease the virulence of the bacterium dramatically. A bacterium may become phage-resistant also by lysogeny (see above), whereby the bacterium becomes immune to the phage and to its close relatives, or by acquiring horizontally a restriction-modification system that degrades the injected phage nucleic acid. In addition, phage resistance may be caused by a mutation in a gene the product of which is essential for the phage replication or assembly. Prerequisites for phage therapy Levin and Bull (2004) suggest that phage therapy only needs to decrease the numbers of infecting bacteria to a level where the host defenses can take care of the remaining bacteria. They also write that it is difficult to understand why a phage that replicates extremely well in the target bacteria fails when it is used for treatment. Understanding this requires a quantitative appreciation of the dynamics of the phage infection process, especially in vivo. Before attempting phage therapy several, sometimes rather demanding, prerequisites should be met: 1. Phage therapy should not be attempted before the biology of the therapeutic phage is well understood. Since the phage–host systems are extremely complicated, this prerequisite has to be faced with some common sense. 2. Phage preparations should meet all the safety requirements; the preparations should be free of bacteria and their components. 7 3. Phage preparations should contain infective phage particles, thus storage of the preparations should be validated. 4. The phage receptor should be known. In a bacterial population of 106–108 bacteria there is a high possibility of spontaneous phage-resistant mutants deficient in the receptor or with an altered receptor. It can be assumed that a mutation eliminating the receptor that functions as a virulence factor of a pathogen (such as LPS) would attenuate the bacterium and then it would be easier for the host immune system to eliminate the bacteria. 5. The efficacy of phage therapy should be tested in an animal model. Each phage may behave differently in vivo. Before a phage as a therapeutic agent can reach the end user many bureaucratic and regulatory hurdles need to be cleared even when the above prerequisites are properly met. Phage (pharmaco)kinetics Surprisingly little detailed research has been performed on the fate of phages in animals and humans. The pharmacokinetics are complicated due to the selfreplicating nature of phage. The in vitro growth data for a phage cannot be directly applied to the in vivo situation, in addition the in vivo data for one phage cannot be transferred to another phage. The use of phages as drugs may differ dramatically from pharmaceuticals/antibiotics due to differences in the phage pharmacokinetics (Payne and Jansen, 2003). Critical parameters that affect phage therapy are the phage adsorption rate, burst size, latent period and initial phage dose, also density-dependent thresholds and associated critical times should be considered (Payne and Jansen, 2001). Another critical parameter is the clearance rate of the phage particles from the body fluids by the reticuloendothelial system. Payne has developed mathematical models to predict the behavior of phages in vivo taking into account population dynamics (Payne and Jansen, 2003). Two paradoxical observations should be mentioned here. It is certain that timing of the phage treatment is critical and that phage administered too early may result in clearing of the phage from the body before it reaches the replication threshold. Similarly, addition of antibiotics in parallel with phage may hamper the phage efficacy. The replication threshold and proliferation threshold are expressions used to describe the situation where a low concentration of host bacteria is present in the culture and it takes some time before the bacteria reach a density where a net increase in phage concentrations ARTICLE IN PRESS 8 M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14 can be seen (Kasman et al., 2002). Apparently, the threshold depends on the rare encounters of the phage with the relatively few host cells. In vivo experiments should demonstrate also whether the physical and chemical conditions encountered in vivo support the phage life cycle. In some body compartments bacteria may reside in an environment that does not provide phages with optimal conditions for infection. Weld et al. (2004) studied mathematical models of phage growth and also tried to fit the formulae to experimental data. They used T4 and K1-5 phages and E. coli strains as host bacteria and rats as the animal model. They concluded that their mathematical model failed to predict phage growth in the rats demonstrating the complexity of phage growth in vivo. Coexistence of the host bacterium and its specific phage was studied by Fischer et al. (2004). They showed that within a bacterial population a fraction of cells may exist in a phageresistant form, thus indicating heterogeneity within the population. Bull et al. (2002) presented an analysis of the now classical Smith and Huggins phage therapy experiments (Smith and Huggins, 1982, 1983; Smith et al., 1987) and repeated some of their experiments. They also introduced and used a resistance competition assay (RCA) and a phage replication assay (PRA) to analyze the parameters involved in the phage treatment. Phages may carry harmful genes A full knowledge of phage genome sequences would be helpful to evaluate possible complications during phage therapy. For example, phages may carry virulence factor or toxin genes (Brüssow et al., 2004; McGrath et al., 2004). Mesyanzhinov et al. (2002) determined the complete nucleotide sequence of Pseudomonas aeruginosa-specific phage fKZ, the largest bacteriophage sequenced thus far (ca. 280 kb with 306 predicted genes), and observed that a number of fKZ gene products has a strong similarity to functionally unknown proteins from a diversity of organisms. This observation must be taken into consideration before the therapeutic use of phages (sequenced or not sequenced) is attempted because some gene products might be toxic for humans. The in vivo phage-associated conversion of Tox! Streptococcus pyogenes into Tox+ bacteria (Broudy and Fischetti, 2003) is an example of the phage-related dangers encountered in nature and it can serve as a warning of the possible side effects of bacteriophage therapy. Included in this category are other phageassociated toxins, of which the CTX cholera toxin (Boyd et al., 2000; Davis et al., 2000; Davis and Waldor, 2003), botulinum toxin (Brüssow et al., 2004; Eklund et al., 1971), shiga-toxin (Beutin et al., 1999; Strauch et al., 2001b, 2004), and diphtheria toxin (Brüssow et al., 2004) are the best known. Phage products Different types of purified bacteriophage products have been evaluated as anti-infective agents. These include bacteriophage lysins and bacteriophage tail-like bacteriocins. One should keep in mind that lysins usually work efficiently only for Gram-positive bacteria. Bacillus anthracis is killed by the g phage lysin PlyG ‘from without’ and PlyG can protect mice infected by B. anthracis (Schuch et al., 2002). Analogously, mureindegrading enzymes (lysozymes, amidases or endopeptidases) of phages infecting Streptococcus pneumoniae have been used successfully in mouse infections (Jado et al., 2003; Loeffler et al., 2001, 2003). A combination of a lysozyme and amidase was found to have a synergistic effect in vitro (Loeffler and Fischetti, 2003) and also in protecting mice (Jado et al., 2003). Additional studies revealed that combinations of a murein-degrading enzyme with conventional antibiotics showed synergistic activities in vitro. However, this has to be confirmed by in vivo studies (Djurkovic et al., 2005). In a mouse model, a single dose of a lysin significantly reduced bacterial colonization by group B streptococci (S. agalactiae) in both the vagina and oropharynx (Cheng et al., 2005). Phage tail-like bacteriocins is another group of phage products able to eliminate target bacteria ‘from without’. This special group of bacteriocins consists of highmolecular-weight particles composed of fragments of bacteriophages and they are produced by a number of Enterobacteriaceae and other Gram-negative bacteria (Bradley, 1967; Daw and Falkiner, 1996). Recently, a phage tail-like bacteriocin, enterocoliticin, was isolated from the culture supernatant of the Yersinia enterocolitica strain 29930. Upon contact with susceptible Yersinia strains the phage tails contract and kill the bacterial cell by forming pores in the cell wall leading to a rapid loss of ions (Strauch et al., 2001a). The cell wall receptor of enterocoliticin was identified as the outer core hexasaccharide of the LPS present in certain serotypes of Y. enterocolitica including serotype O:3 (Skurnik et al., 1995, 1999; Strauch et al., 2003). The efficacy of enterocoliticin as an antimicrobial agent against infections with pathogenic Y. enterocolitica O:3 strains was assessed recently (Damasko et al., 2005). In cell cultures exposed to Y. enterocolitica enterocoliticin killed bacteria adhering to the surface of eukaryotic cells whereas intracellularly located bacteria were not accessible to the bacteriocin. In mice infected orally, an increase in the Y. enterocolitica numbers was inhibited at time points shortly after the oral application of ARTICLE IN PRESS M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14 enterocoliticin indicating that the particles were effective on recently introduced pathogens. Repeated application of enterocoliticin, however, did not prevent the colonization of the gastrointestinal tract by Y. enterocolitica suggesting that the bacteria rapidly escaped out of the reach of the enterocoliticin in vivo. On the other hand, enterocoliticin survived well in the hostile environment of stomach juices. Use of phage tail-like bacteriocins as antimicrobial agents most likely will be limited only to in vitro applications. In this section could also be discussed the work of Hagens et al. (2004) who describe the use of nonreplicating genetically modified phage Pf3R to kill P. aeruginosa in a mouse model. They deprived the filamentous phage Pf3 genome of the export protein gene and replaced it with the restriction endonuclease BglIIR gene. In this way the phage infects the host bacteria expresses the restriction endonuclease BglII that breaks the host genomic DNA but not that of the phage (which is missing the recognition site) and kills the host. In vitro Pf3R released only small amounts of endotoxin from the bacteria when compared to a lytic phage. In vivo experiments with infected mice also demonstrated a protective effect against 3–5 times the median lethal dose of bacteria (Hagens et al., 2004). Recent in vivo phage studies In a recent paper from Brüssow and colleagues (Chibani-Chennoufi et al., 2004) systematical experiments with E. coli phages were conducted. A collection of E. coli-specific phages were isolated from stool samples of children in Bangladesh, environmental water in Bangladesh and sewage in Switzerland. These phages were then tested against a collection of E. coli strains, both pathogenic (EPEC and EHEC) and non-pathogenic, the latter representing the ECOR collection (Ochman and Selander, 1984). Altogether four phages related to the well characterized T4 coliphage that showed the widest and complementary infection range among the strains were selected for the mouse experiments. The endogenous E. coli gut flora of ten conventional mice was assayed for phage susceptibility. Altogether 500 lactose-positive colonies were tested. The results indicated that practically all E. coli cells were lysed by at least one of the phages. The susceptibility of the E. coli to the phages was then determined in vivo by applying orally increasing doses of phages mixed in the drinking water. At different time points, lactose-positive colonies were counted from the stools, however, only a slight decrease in the numbers was seen (counts dropped from 106.2 to 105.7). The recovered colonies were sensitive to phage lysis and the phages survived the passage through 9 the gut although they did not multiply significantly. Thus the survival of the bacteria in the gut during the phage passage could only be explained by some physiological reasons that prevented phage-induced lysis. The authors went further and used axenic mice that were infected with a single E. coli strain and then were given phages in the drinking water. In these mice the phage titers in the stools increased in one day from the 105/ml in the drinking water to 1010/ml in the stool, at the same time numbers of E. coli in the stools dropped from 108 to 104, from where the numbers leveled off during the subsequent days to 105. Again the recovered colonies were sensitive to the phages suggesting that they have resided in gut sites protected from phage. Paisano et al. (2004) infected human dental roots with Enterococcus faecalis to study the antimicrobial effect of bacteriophages. They could show reduction of viability of the bacteria in root canals when different phage:bacteria ratios were used. In a very thorough study, Biswas et al. (2002) performed experiments using a mouse model of vancomycin-resistant Enterococcus faecium (VRE) infection. They isolated VRE-specific phages from raw sewage and selected one that showed an antibacterial effect against 79% of the studied strains in vitro for animal experiments. They showed first that intraperitoneally (i.p.) 45 min post infection administered phage was able to rescue mice from VRE bacteremia and that the rescue was associated with a significant decrease in bacterial numbers in blood. They also demonstrated that phage administration before 5 h post infection still fully rescued the mice while treatment delayed beyond 5 h rescued only some of the mice (Biswas et al., 2002). Bacteriophage fMR11 was tested in mouse infections caused by Staphylococcus aureus (Matsuzaki et al., 2003). The bacteriophage was isolated after induction of S. aureus strains by mitomycin. It was selected for experimental studies due to its broad ability to infect and lyse the 75 S. aureus indicator strains. The 43-kb phage genome was completely sequenced and did not carry any known toxin, virulence factor or antibiotic resistance genes. The authors also characterized the phage with regard to its biological features. Mice were infected i.p. with an S. aureus dose that killed 80% of the untreated mice within 24 h and all within 7 d. Most of the mice died between 6 and 7 h after infection. Mice receiving phage i.p. immediately following the bacterial challenge were protected. In an experimental design where mice were infected with the fMR11 lysogen strain no protection was observed allowing the authors to conclude that a direct bactericidal effect of the phage was the principal determinant of the protective effect and not any indirect effect such as a phage-stimulated immune response (e.g. production of cytokines) (Matsuzaki et al., 2003). Phage could rescue mice when administered 60 min later than the bacteria. Phage and ARTICLE IN PRESS 10 M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14 bacterial numbers in the circulation were determined after the infection and showed that the bacterial load was much lower in the blood of phage-treated mice when compared to those that received only bacteria. They also noticed that phage titers in mice infected with bacteria remained higher than the titers in mice that received only the phage. This suggested that the phage replicated in the infected mice and consumed the bacteria. This happened until the bacteria disappeared from the circulation which was paralleled by elimination of the phage (Matsuzaki et al., 2003). As an example of the failure in phage therapy we will briefly describe our own phage therapy experiments that were performed with Y. enterocolitica O:3 strains. Strains of this serotype are low-pathogenicity strains of Y. enterocolitica and cause only a mild diarrhea in mice. Phage treatments were designed to test the possibility of eradicating selectively pathogenic bacteria from the gut microflora in healthy mice. Such an application might be attractive to combat Y. enterocolitica O:3 in pigs which is the main source of human infections. Y. enterocolitica serotype O:3 strains 6471/76 (Skurnik, 1984) and 13169 (Strauch et al., 2001a) were selected for phage challenge studies. Eight-week-old female BALBc mice were used in the experiments, phages and strains were applied orally with a feeding syringe in a volume of 0.5 ml per mouse. Yersinia phage PY100 was isolated from pig manure collected from a farm in Germany and chosen for the experiments, as it formed in vitro large clear plaques at 37 1C on cultures of Y. enterocolitica O:3. Phages were administered orally in a single application with a dose of 4 " 1010 pfu to study the dissemination of phage particles in the mice. Five mice were killed after 6, 12, 24, and 48 h, and PY100 titres (pfu/g) were determined in the following samples: ileum, caecum, faeces, spleen, liver, and kidney. It was found that the phage particles were active for at least 24 h within the gastrointestinal tract (titres between 104 and 106 pfu/g of organ and faeces, respectively). In some mice, phage particles were even found in deeper organs indicating that particles had passed the gastrointestinal barrier. Since a single application of PY100 did not inhibit the colonization of mice by Y. enterocolitica O:3 strain 13169, the experiment was repeated by administering the phage at 24 h intervals four times starting 1 h after the infection (Fig. 1; Kaspar, 2003). Mice were killed after 24, 48, 72, 96, and 170 h (five mice per time point). In Fig. 1 the recovered Yersinia cfu are shown: mice killed after 24 h had received one phage dose, mice sacrificed after 72 h had received three phage doses and mice sacrificed after 170 h four doses. There was no significant difference between mice treated with PY100 phage and untreated mice. Yersinia titres were in the range from 106 to 108 cfu/g of tissue or faeces. Fig. 1. Recovery of Y. enterocolitica O:3 13169 bacteria in organs and faeces of mice after repeated application of phage PY100. Each animal was infected with 1.8 " 109 cfu of strain 13169; each phage dose contained 1.85 " 1010 pfu per animal. (!) control: only Yersinia infection; (+) mice treated with phages. Bars represent mean cfu (5 animals per group), standard deviation is indicated. Another experiment on a smaller scale was carried out with yersiniophage fYeO3–12 (Pajunen et al., 2000, 2001) as a protective phage. After oral administration phage fYeO3–12 was also active in the gastrointestinal tract for more than 24 h. Mice were infected with Y. enterocolitica strain 6471/76. Each mouse was infected orally with 1.9 " 109 cfu. In the control group, mice were infected with Y. enterocolitica only. In the therapy group all mice received phage (a dose of 5.5 " 108 pfu of fYeO3–12) 1 h after the bacterial challenge and additional doses after 24, 48 and 72 h. After 6, 24, 48, 72, 96, and 120 h two mice in each group were killed and investigated for Y. enterocolitica O:3 (cfu/g) and fYeO3–12 (pfu/g, only for therapy group). The results obtained with phage fYeO3–12 were similar to those of PY100, as untreated and treated animals yielded approximately the same Yersinia titres in the gastrointestinal tract (titres between 105 and 108 cfu, Strauch and Skurnik, unpublished data). The major finding of our experiments was that phage treatment was not able to eradicate the bacteria from the mice even though in in vitro cultures the bacteria were efficiently killed. In summary, these phage therapy trials with Y. enterocolitica O:3 demonstrated that Y. enterocolitica colonization in mice cannot be controlled by phage therapy, at least with the regimen used by ourselves. It would not be surprising if similar results are obtained with other bacterial pathogens. Phage therapy trials in chickens Huff and colleagues have explored the possibilities of phage therapy using chicken challenged with a respiratory ARTICLE IN PRESS M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14 infection-causing Escherichia coli. Their first paper reported just the clinical signs of the infected birds, without analysis of the phage or bacterial loads (Huff et al., 2002a, b). Phage mixed with E. coli prior to an air sac infection protected chicken but phage administered into the drinking water did not. In their second study they used an aerosol spray of phage instead of phage in the drinking water and concluded after observing some protection against the infection that an aerosol spray was a promising way of administration of the phage (Huff et al., 2002a, b). In their third study an aerosol spray and intramuscular injection of bacteriophage were compared (Huff et al., 2003b). Phage given as an aerosol spray immediately after the E. coli infection but not after 24 h was protective, whilst the intramuscular phage protected also when given immediately, 24 or 48 h after challenge. Presence of phage in the blood was followed. Almost no birds had phage in the blood after aerosol spraying whilst high numbers were present after intramuscular administration (Huff et al., 2003b). Multiple intramuscular treatments proved to be more effective than a single dose (Huff et al., 2003a). Another clinical trial was conducted to evaluate a combination of the antibiotic enrofloxacin and intramuscularly administered bacteriophage (Huff et al., 2004). Both treatments individually provided effective treatments of the E. coli infection, but the synergy between the two treatments led to a total protection of the birds, thus suggesting a significant value of the combined treatment. Phage therapy in fish aquacultures A Japanese group has studied the potential to use phage to prevent fish infections caused by Lactococcus garviae or Pseudomonas plecoglossicida (Park and Nakai, 2003; Park et al., 2000). Phages were administered either i.p. or orally as phage-impregnated feed. The authors also presented the idea of introducing the phage inside bacteria to fish, and found no difference in protective efficacy between them. Phages riding inside a Trojan horse The escape of invasive pathogens into closed tissue and organ compartments may block the effective use of bacteriophages especially if the phage cannot actively follow the bacteria. Replicating phages may enter the compartment inside an invading bacterium analogous to the Trojan horse. It is however quite clear that many variations of this theme will be encountered among different bacteriophage/pathogen systems. In an attempt to utilize the Trojan horse concept, Broxmeyer et al. (2002) developed a system where Mycobacterium smeg- 11 matis, an avirulent mycobacterium, was used to deliver the lytic phage TM4 into macrophages in tissue cultures where both M. avium and M. tuberculosis resided. The results showed that the treatment was able to reduce the numbers of viable intracellular bacilli (Broxmeyer et al., 2002). Conclusions Based on the few examples presented here, the use of bacteriophages to control bacterial infections shows therapeutic promise. The worldwide increase of pathogenic bacteria resistant to antibiotics makes it an imperative to exploit alternative strategies to combat this threat. The therapeutic use of bacteriophages – perhaps in combination with antibiotics – may turn out to a valuable approach. However, it is also quite clear that the safe and controlled use of phage therapy will require detailed information on the properties and behavior of the specific phage–bacterium system, both in vitro and especially in vivo. The in vivo susceptibility of bacterial pathogens to bacteriophages is still largely poorly understood. References Alisky, J., Iczkowski, K., Rapoport, A., Troitsky, N., 1998. Bacteriophages show promise as antimicrobial agents. J. 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