International Journal of Antimicrobial Agents 30 (2007) 118128

Review

Bacteriophages: an appraisal of their role in the treatment of bacterial infections

Geoffrey William Hanlon
School of Pharmacy and Biomolecular Sciences, University of Brighton, Moulsecoomb, Brighton BN2 4GJ, UK

Abstract Bacteriophages were rst used successfully to treat bacterial infections a decade before penicillin was discovered. However, the excitement that greeted those initial successes was short-lived, as a lack of understanding of basic phage biology subsequently led to a catalogue of clinical failures. As a consequence, bacteriophage therapy was largely abandoned in the West in favour of the newly emerging antibiotics. Now, as the problem of antibiotic resistance becomes ever more acute, a number of scientists and clinicians are looking again at bacteriophages as a therapeutic option in the treatment of bacterial infections. The chances of success second time round would appear to be much better given our current extensive knowledge of bacteriophage biology following their important role in underpinning the advances in molecular biology. We also have available to us the experience of nearly 80 years of clinical usage in the countries of the former Soviet Union and Eastern Europe as well as a political climate that encourages sharing of that knowledge. This review outlines those features of bacteriophages that contribute to their utility in therapy and explores the potential for their re-introduction into Western medicine. An abundance of clinical evidence is available in the Soviet literature but much of this is technically awed and a more realistic appraisal of the clinical value of phages can be obtained from animal studies conducted in the West. As interest in bacteriophages increases, a number of companies throughout the world have begun investing in phage technology and this has led to novel approaches to therapy, some of which will be discussed. 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Keywords: Bacteriophages; Phage therapy; Bacterial infections

1. Introduction The emergence of profoundly antibiotic-resistant pathogens such as Mycobacterium tuberculosis, Enterococcus faecalis, Staphylococcus aureus, Acinetobacter baumannii and Pseudomonas aeruginosa is a cause of major concern. It has been stated that although the main focus of public and media attention has been methicillin-resistant S. aureus (MRSA), we are closer to the end of the antibiotic era with Gram-negative pathogens such as A. baumannii [1]. Equally worrying is the fact that new antibiotics are not being developed at a rate sufcient to replace those drugs that are becoming less useful. Spellberg et al. [2] surveyed the Research and Development (R&D) programmes of the 15 largest multinational pharmaceutical companies and found that only ve antibacterial agents were currently undergo

ing development, none of which represented new classes of antibiotics. It is therefore apparent that the prospect of any novel antibiotics being available for clinical use in the next decade is low. This has become the driver in the search for alternative strategies to treat infections caused by antibioticresistant bacterial pathogens. The remainder of this article will review the current potential of a form of therapy that pre-dates antibiotics, i.e. the use of bacteriophages in the treatment of bacterial infections [3].

2. Bacteriophage biology and interactions with host bacteria Bacteriophages (phages) are bacterial viruses that play a profound role in the evolution of their host. Whole genome sequencing of bacteria has revealed that phage elements contribute signicantly to sequence diversity and can potentially inuence pathogenicity. Phages are ten times more numer-

Tel.: +44 1273 642 082. E-mail address: g.w.hanlon@brighton.ac.uk.

0924-8579/$ see front matter 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2007.04.006

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Fig. 1. Diagrammatic representation of a typical bacteriophage.

ous in the environment than bacteria, making them the most abundant life forms on Earth, with an estimated 1032 bacteriophages on the planet [4]. They are most frequently isolated from aquatic environments but are generally found wherever bacteria reside and have co-evolved with bacteria over the 34 billion years during which life has existed on Earth. It is certain that the variety of phages that have been isolated and characterised represent only a tiny fraction of the total. Bacteriophages cannot infect mammalian cells but specifically target bacteria. This specicity is highly rened and each phage will only attack one species or in some cases a single strain of bacterium. There are a variety of different morphological types of bacteriophage, although the majority exhibit the characteristics shown in Fig. 1. The head (or capsid) is a protein shell often in the shape of an icosahedron; this contains the viral genome that usually comprises double-strand (ds) DNA. The tail may or may not be a contractile structure and to this are connected usually six tail bres containing receptors at their tips that recognise attachment sites on the bacterial cell surface. Not all phages possess tails or tail bres and here other attachment mechanisms are in place. Most of the bacteriophages of relevance to this review belong to three families (see Table 1), the Siphoviridae, the
Table 1 Description of bacteriophage families Family name Corticoviridae Cystoviridae Fuselloviridae Inoviridae Leviviridae Lipothrixviridae Microviridae Myoviridae Plasmaviridae Podoviridae Rudiviridae Siphoviridae Tectiviridae Morphology Icosahedral capsid with lipid layer Enveloped, icosahedral capsid, lipids Pleomorphic, envelope, lipids, no capsid Rod-shaped with helical symmetry Quasi-icosahedral capsid Enveloped laments, lipids Icosahedral capsid Contractile tail Pleomorphic, envelope, lipids, no capsid Short, non-contractile tail Helical rods Long, non-contractile tail Icosahedral capsid with inner lipoprotein vesicle Genome dsDNA dsRNA dsDNA ssDNA ssRNA dsDNA ssDNA dsDNA dsDNA dsDNA dsDNA dsDNA dsDNA

ds: double-stranded; ss: single-stranded.

Myoviridae and the Podoviridae [5], comprising 15 genera. The remaining phages occupy 10 families each with a small number of members. Bacteriophages can exhibit one of two types of life cycle: virulent or temperate [6]. Virulent phages bring about rapid lysis and death of the host bacterial cell, whereas temperate phages spend part of their life cycle in a quiescent state called prophage. In the lysogenic cycle, viral DNA is often integrated into the host cell DNA but may also exist as a plasmid. Prophage DNA will be replicated when the host cell genome replicates and so daughter cells will inherit the viral DNA. For the purposes of phage therapy, temperate phages have little value. The life cycle of a typical lytic bacteriophage is shown in Fig. 2. The virus encounters its bacterial host during random motion and attaches via specic receptor sites that may be any one of a wide variety of cell surface components, including protein, oligosaccharide, teichoic acid, peptidoglycan and lipopolysaccharide [6]. In some cases the attachment sites might be present on the cell capsule, agella or even conjugative pili. Initially the attachment is reversible but then becomes irreversible and is followed by transfer of phage genetic material into the host cell. Injection of the phage genome into the bacterial cell again can occur by a variety of mechanisms depending on the morphology of the virus but often involves contraction of the tail and formation of a hole within the bacterial cell wall. Many of the bases present on the phage DNA are chemically modied to confer protection against attack by cellular restriction and nuclease enzymes. The viral genome is then transcribed by host cell RNA polymerase, producing early mRNA that has the effect of taking over the metabolic machinery of the bacterium, redirecting its metabolic processes to the manufacture of new virus components. These components are then assembled into complete virions. Following construction and assembly of new phage particles within the host cell there remains the problem of release into the environment. Nearly all dsDNA phages have developed enzymes that attack the bacterial peptidoglycan, which can be lysozymes that target sugar bonds, endopeptidases that target peptide linkages or amidases that act on amide bonds [7]. These lytic enzymes, generically termed muralytic enzymes or endolysins, are produced within the cytoplasm and require another enzyme to enable them to cross the cytoplasmic membrane to reach their substrate. This enzyme is a holin that disrupts the membrane allowing the lysin to degrade the peptidoglycan [7,8]. In this way the holin controls the timing of cell lysis and release of phage progeny. Some phages have a lamentous morphology and these can escape from the host cell by extrusion through the cell wall without causing destruction of the host. These phages are of no relevance to therapy and will not be considered further. The period of time from adsorption to the lysing of the host cell and release of viral progeny is referred to as the latent period. Infective phages are, however, present within the cell before lysis and can be released from the cell using

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Fig. 2. Lytic cycle of a bacteriophage.

chloroform. The period of time from adsorption to the rst appearance of chloroform-induced phage is called the eclipse period. From a single bacterial cell, in excess of 100 new virus particles may be liberated and each of these is able to go on to infect a new bacterial cell. This process of phage replication was originally described by Ellis and Delbruck [9] and is referred to as the single-step growth curve. The infection cycles have the potential to continue until all susceptible bacterial cells have been killed. Temperate phages are viruses that do not automatically enter a lytic cycle but instead integrate their DNA into the host cell DNA. The bacterial cells are then termed lysogenic. When the bacterial DNA replicates, the phage DNA replicates at the same time and so each daughter cell will contain the viral DNA (known as prophage). Cells may undergo several rounds of division but occasionally one will spontaneously lyse and liberate progeny phage. Alternatively, a population of lysogenic cells may be induced to lyse by subjecting them to stress such as treatment with mutagenic agents or exposure to ultraviolet light. Some temperate phages such as Mu can switch between lysogeny and lytic growth under the inuence of high temperature and stationary phase [10]. The prophage directs the synthesis of a repressor protein that blocks the transcription of its own genes and also those of closely related bacteriophages. The presence of a prophage can therefore confer upon a bacterial cell some sort of immunity to infection by other phages. Lysogenic bacteria may possess other advantages in terms of the acquisition of genes conferring pathogenicity

or increased virulence. When a prophage escapes regulation by the repressor, its DNA is cut free allowing it to embark upon a lytic cycle. However, excision of prophage DNA is often imprecise and bacterial genes adjacent to the prophage DNA may be incorporated into the infectious phage DNA and then transferred to subsequent host cells. This process is known as transduction and is responsible for the horizontal transfer of genes from one bacterial cell to another. Examples of virulence genes include those for host attachment, invasion and survival as well as the production of toxins. Important toxin genes known to have been acquired by transduction include the neurotoxin of Clostridium botulinum, the diphtheria toxin of Corynebacterium diphtheriae, the Shiga toxins found in Escherichia coli O157 and the cholera toxin in Vibrio cholerae [11]. Furthermore, the advent of whole genome sequencing of bacteria has revealed that prophage or phage-like elements are abundant and not only contribute to sequence diversity but have clearly played a signicant role in evolution [12].

3. Clinical experience of bacteriophages as therapeutic agents There have been numerous excellent reviews detailing the fascinating history of the discovery of bacteriophages, early attempts at employing them for the purposes of antibacterial therapy and their subsequent decline [3,1315]. Readers

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interested in the details of this period are referred to these publications and references therein. It is of relevance to note here that in Eastern Europe and the former Soviet Union bacteriophages have been used clinically for the treatment of bacterial infections continuously since 1919. In the West, however, they have not been taken seriously since the advent of antibiotics. A major reason for the demise of phage therapy in the West was a great deal of poor science that was performed in the early days as a result of lack of knowledge of bacteriophage biology. We now know a great deal about the biology of bacteriophages that have been instrumental in improving our understanding of molecular genetics and basic cell biology and are therefore well placed to begin to revisit some of the past work and to re-evaluate the potential of phage therapy. It is easy to see why there is a move in favour of bacteriophages as therapeutic agents, as they appear to offer a number of advantages over the use of antibiotics. Bacteriophages target only the pathogens of interest and the normal gut microora are not affected. A precise diagnosis must be obtained in advance of therapy and empirical treatments are not possible, but problems of disturbance of normal bowel ora and potential overgrowth of secondary pathogens are avoided. Their mechanism of action is completely different from all available antibiotics and so they are effective even against bacteria exhibiting multiple antibiotic resistance. Hence, even if they are not used as rst-line therapy, bacteriophages represent a very useful last line of defence. The pharmacokinetics of bacteriophage therapy is such that the initial dose increases exponentially as the virus multiplies within the susceptible bacterial host and is subsequently released. Often there is no need to carry out repeat dosing. There is evidence that phage can penetrate poorly vascularised tissues and can cross the bloodbrain barrier [16]. Extensive clinical experience in the former Soviet Union and Eastern Europe has revealed very few cases of side effects or allergic reactions. It is less certain what immunological effects might arise on multiple dosing. Bacteriophages are at rst sight cheap and easy to produce. However, it is by no means a trivial exercise to develop the highly virulent, lytic, broad-spectrum, non-transducing phage appropriate for effective therapy. The main focus for phage therapy R&D has been the Eliava Institute of Bacteriophage, Microbiology and Virology, located in Tbilisi, which is now the capital of the independent Republic of Georgia, although formerly it was part of the USSR. The Eliava Institute became both a major R&D establishment and also the principal manufacturer of phage products to the Soviet Union. At the height of its activity the Institute employed over 1000 people and produced tons of phage products daily both for therapy and

prophylaxis throughout the Soviet Union. A major user of phage products was the military and much of the literature on phage therapy during that period centres on its use in diseases such as gangrene and dysentery, both of which were a major problem in wartime. A major contribution of the Eliava Institute has been the development of techniques for the isolation, purication, screening and selection of highly virulent, lytic bacteriophages appropriate for clinical use. An abundance of experience, knowledge and expertise therefore exists within the former Soviet Union generally, and specically within the Republic of Georgia, on the application of bacteriophages in the treatment of bacterial infections. However, much of the clinical work has been published in local journals often written in Georgian or Ukrainian and hence the majority of it has not reached the West. However, those articles that have been seen received criticism for their poor experimental design, lack of detailed information and lack of proper controls. Chanishvili et al. [17] have reviewed the old Soviet literature and conclude that, despite some of these shortcomings, phage therapy has a proven clinical track record and its potential should be re-evaluated. Within Eastern Europe the principal centre for R&D activity was the Institute of Immunology and Experimental Therapy, which was founded in 1952 in Wroclaw, Poland. Workers from that centre have recently summarised a large amount of clinical data emanating from Poland during the 1980s in a series of papers [1825]. Slopek and co-workers described the results obtained between 1981 and 1986 for a total of 550 patients (age range 1 week to 86 years) treated at 10 different hospitals. Of these patients, 518 had previously been unsuccessfully treated with antibiotics. Their conditions ranged from wound infections, respiratory tract infections to peritonitis and bacteraemia, whilst the pathogens included staphylococci, Klebsiella, Pseudomonas, E. coli and Salmonella. The phages were obtained from a culture collection of bacteriophages and treatment involved 10 mL of suspension by mouth half an hour before meals (after gastric acid neutralisation) or topical application using phage-soaked dressings. The success rate, dened by complete recovery plus negative cultures, was reported as ranging from 75% to 100% (94% overall) depending upon the pathogen. Cislo et al. [25] treated 31 patients with chronic suppurative skin infections using phages from the collection of the Institute applied as moist applications or by mouth. Infecting pathogens were described only as Staphylococcus, Pseudomonas, Klebsiella, E. coli and Proteus. Results were based on a four-point scale and the authors reported an outstanding therapeutic effect in 16 cases and a marked improvement in 6 cases. In seven patients the treatment was terminated owing either to lack of improvements or to side effects. Although on the face of it the results appear promising, the lack of control groups again makes proper evaluation difcult.

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4. Animal studies investigating the potential of phage therapy As researchers in the West became aware of the work being conducted in the Soviet Union and Eastern Europe, some decided to investigate the value of phage therapy using much more robust experimental protocols. Smith and Huggins conducted a series of extremely well-designed experiments evaluating the use of bacteriophage in experimental infections of animals [2630]. In their rst paper [26], mice were infected with a toxigenic, encapsulated strain of E. coli isolated from a baby with meningitis. The bacteriophage used was specic for the K1 capsular antigen and a single intramuscular (i.m.) dose was found to be as effective in controlling infection as multiple i.m. doses of tetracycline, ampicillin, chloramphenicol or trimethoprim + sulfafurazole. The use of an anti-K1 phage had the advantage not only of being much more effective than other phages, but it also meant that any resistant bacterial mutants which emerged were K1-negative and hence much less virulent. Subsequent work by these authors included the treatment of E. coli diarrhoea in calves, piglets and lambs [27], prophylaxis of diarrhoea in calves [30] and an investigation into the effect of environmental conditions on the survival of therapeutic bacteriophages [29]. The favourable results obtained in each of these studies provided robust, repeatable evidence that bacteriophage therapy is effective in treating infections in animals and may have a place in the treatment of infections in humans [31]. This theme was taken up by Soothill and co-workers who explored the effectiveness of phage in controlling infections caused by P. aeruginosa and A. baumannii in burn wounds [32,33]. These pathogens are particularly problematic in these cases since both species exhibit multiple resistance to antibiotics, can spread to cause generalised infections and can lead to rejection of skin grafts. Initially using guinea pigs as a model system, Soothill showed that the destruction of skin grafts by P. aeruginosa could be prevented by prophylactic administration of phage [32]. Phages were also shown to give protection against systemic infections caused by these pathogens in a mouse model. Using an Acinetobacter phage dose as low as 100 plaque-forming units (PFU) protected mice against a dose of 108 colony-forming units (CFU) of A. baumannii, which was ve times the 50% lethal dose (LD50 ) and it was observed that phage multiplication had occurred in vivo. Favourable results were also obtained with P. aeruginosa but a higher dose of phage was required. The work by Soothill and co-workers thus provided further evidence of the potential of phage therapy but also gave insights into areas that needed to be addressed. More recently, Tanji et al. [34,35] have investigated the use of orally administered bacteriophages to control E. coli O157:H7 in the gastrointestinal tracts of mice. The phages were obtained from animal faeces and human sewage, and three highly virulent phages with the broadest spectrum of activity were selected to produce a therapeutic cocktail. This mixture proved to be effective in eliminating E. coli O157:H7

from aerobic batch culture in vitro, and repeated oral administration eliminated this organism from the gastrointestinal tract of infected mice. However, these phages also had lytic activity against non-O157 strains of E. coli. Other workers have isolated bacteriophages that act specically against E. coli O157 serotypes and not non-O157 E. coli, but these appeared to be less useful as the high multiplicity of infection required to be effective (103 PFU/CFU) and the lack of evidence of viral replication suggests that death may be due to lysis from without [36]. It is evident from the above that most of the animal work has been conducted on Gram-negative bacteria and less has been published on the effect of bacteriophages on Gram-positive pathogens. Biswas et al. [37] explored the use of bacteriophage to rescue bacteraemic mice infected with vancomycin-resistant Enterococcus faecium (VRE). This bacterium is particularly problematic in terms of its antibiotic resistance and there have been suggestions of resistant strains emerging even to the newer antibiotics such as linezolid and quinupristin/dalfopristin. Hence, there is a powerful argument for the exploration of alternative therapeutic options. Animals administered with 109 CFU of VRE by intraperitoneal (i.p.) injection were all dead within 48 h. When administration of VRE was followed after 45 min by an i.p. injection of 3 108 PFU of the monoclonal phage preparation, all the animals survived. The phage preparation was 50% effective even when the treatment was delayed up to 24 h, at which point the animals were nearly at the point of death. An important observation with this study was that the effect was due to the bacteriophage infection process itself and not to a non-specic immune response. As the principle of bacteriophage therapy has become accepted, a number of groups have explored ways in which phages may be modied to optimise their therapeutic efciency. A report by Geier et al. [38] showed that bacteriophages injected into germ-free mice were rapidly eliminated from the circulation by the reticuloendothelial system (RES) even in the absence of an antibody response. This preliminary nding was reinforced in a later paper by Merril et al. [39] who showed that 1011 PFU of phage injected intraperitoneally into a mouse had declined to 102 PFU at 48 h and to undetectable levels after 120 h. The purpose of this study was to serially passage surviving phage through the animal model and to obtain strains that had an enhanced ability to avoid the RES. This proved successful, as two phage isolates were obtained that exhibited survival at 18 h post injection 16,000 and 13,000 times greater than the parental strain. The release of endotoxin from Gram-negative bacteria causes signicant morbidity and mortality in those patients with sepsis and in some instances treatment with lytic antibiotics such as the -lactams may exacerbate the situation. Certain studies have indicated that antibiotics that bind to penicillin-binding protein 2 (PBP2), such as imipenem, give rise to less endotoxin release than antibiotics binding to PBP3, such as ceftazidime [40]. However, recent clinical studies have suggested that these antibiotics had similar

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effects on endotoxin and subsequent cytokine release during the treatment of Gram-negative infections [41,42]. Despite uncertainty over the precise role of antibiotics in endotoxin release [43,44] it has been supposed that virulent bacteriophages by their very nature will cause profound liberation of endotoxin during therapy. The use of non-lytic temperate phages for therapy is of course not an option, but Matsuda et al. [45] have explored the potential of lysis-decient bacteriophages in the treatment of mouse peritonitis. They used a lysis-decient E. coli bacteriophage (t amber A3 T4) with a mutation in the gene for the production of holin. This was a genetically engineered mutant of T4 phage only able to infect E. coli. Hence these phages were able to infect host cells and to replicate within but not to lyse. The bacterial cells were thus killed but remained largely intact following infection. When used in an experimental murine peritonitis model, treatment with lysis-decient phages was shown to improve survival of the animals compared with other treatment modalities. In addition, these phages killed the infecting bacteria but in a manner that signicantly minimised the release of endotoxin and inammatory cytokines compared with treatment using lytic phages or the -lactam antibiotic latamoxef [45].

5. Bacteriophage preparations Experimental phages are frequently isolated from environmental sources and undergo minimal optimisation procedures before use. However, to be successful clinically, bacteriophages must undergo rigorous selection and characterisation. Procedures for isolating and purifying phages from the environment are described in detail by Carlson [46] and the reader is referred to this text for further information. Jassim et al. [47] have developed a phage breeding protocol to isolate phages possessing increased virulence within a population. This process was based upon the use of an antiviral compound derived from pomegranate extract that destroyed bacteriophages free in suspension but not if bound to a host cell surface. When added to broth containing both phage and host bacteria, the process will select those phages that adsorb most rapidly, as the unbound virus will be killed. The progeny from that infection will be passed through repeated rounds of breeding to produce virus with enhanced virulence. This technology was employed to produce a bacteriophage that was specic for L-forms of Listeria monocytogenes [48]. The Eliava Institute in Tbilisi, Republic of Georgia, has developed its bacteriophage collection over many decades and currently produces a range of phage preparations in a variety of pharmaceutical forms that are able to be administered topically, orally, rectally, by inhalation or by injection. They have been fully characterised and are all highly virulent, are not susceptible to host restriction and do not carry any virulence genes. The bacteriophage preparation may be a single clone (monoclonal) active against one species of bac-

terium (monovalent) or in the form of cocktails of phage that are able to be used against a broad range of pathogens. One of their most important phages is a highly virulent, monoclonal staphylococcal bacteriophage active against 8095% of S. aureus strains, including MRSA. This product can be used for local and generalised infections, including sepsis in the newborn, osteomyelitis, wound infections, pneumonia, etc. This phage represents an ideal agent from a regulatory standpoint in that it has been fully characterised both biologically and genetically and is able to be used on a stand-alone basis. Pyophage is the commercial name given to a cocktail of phages active against staphylococci, streptococci, P. aeruginosa, Proteus spp. and E. coli that can be used for the treatment and prophylaxis of purulent wound infections. It is also used in surgery (both pre and post operatively), burn wounds, osteomyelitis, skin infections, and eye and ear infections. This phage mixture is also used in a commercial wound dressing product called PhagoBioDerm [49], which is a novel biodegradable polymer based on poly(ester amide)s impregnated both with the phage cocktail and the antibiotic ciprooxacin. The dressing has been used successfully to treat infected wounds including those containing multiply resistant S. aureus [49,50]. The Institute has a long history of treating gastrointestinal infections and has developed an 11-component mixture active against six different species of Salmonella as well as a 17component cocktail effective against a broad range of gut pathogens.

6. Mycovirus The ubiquitous nature of bacteriophages makes it reasonable to suggest that fungi may also be susceptible to naturally occurring viruses and that these could form the basis of therapy of systemic and other mycoses. In fact, fungal viruses (or mycoviruses) are widespread but are usually associated with asymptomatic, persistent infections and only a few cause variable phenotypic effects [51]. These viruses typically possess a genome containing dsRNA that is present in different numbers of segments [52]. Two families, the Totiviridae and the Partitiviridae, consist of non-enveloped isometric particles that typically cause latent infections. Viruses belonging to the Hypoviridae can give rise to considerable morphological and physiological changes in the host organism. dsRNA viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents [53]. In most cases the virus is located in the cytoplasm of the cell but some have been found associated with the mitochondria. They generally do not have an extracellular phase in the same way as lytic bacteriophages. Transmission of the virus is usually vertical from parent to progeny, and interspecies transfer has only occasionally been reported by protoplast fusion. A recent study by van Diepeningen et al. [54] showed that of 668 examples of black Aspergillus spp. obtained

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from all over the world, 10% were infected with dsRNA mycoviruses. Whilst the viruses themselves may not offer much potential for therapy, some of the mycoviruses encode for killer toxins. Weiler et al. [55] studied the secretion of a 10 kDa protein toxin (zygocin) from Zygosaccharomyces bailii that rapidly killed a broad spectrum of yeasts and fungi including the human pathogens Candida albicans, Candida glabrata and Sporothrix schenckii by disrupting cytoplasmic membrane function. The toxin was encoded by a dsRNA virus that stably persists in the cytoplasm of the yeast. However, not all killer toxins are virus encoded. Some are chromosomally encoded and others are found on plasmids. It is possible that these toxins may form the basis of new forms of therapy for recalcitrant fungal infections, provided their toxicity to mammalian systems is acceptable.

7. Problem therapeutic areas 7.1. Bacteriophage control of biolm infections Whilst there are signicant therapeutic problems posed by bacterial pathogens that are intrinsically resistant to antibiotics, infections caused by bacteria that develop phenotypic resistance as a result of their growth environment are also an issue. One major route by which this may arise is the formation of bacterial biolms adherent to surfaces, in particular on indwelling medical devices [56]. Indeed, this form of growth is far more common in the environment than planktonic growth. The denition of a biolm has evolved over the years, but recently Donlan and Costerton [57] described it as . . . a microbially derived sessile community characterised by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a matrix of extracellular polymeric substances that they have produced, and exhibit an altered phenotype with respect to growth rate and gene transcription. It has been shown that mature bacterial biolms are able to resist antibiotic concentrations up to 1000 times greater than those tolerated by planktonic cells. The mechanisms proposed for this increased resistance centre on observations that: (i) the extracellular polymeric substance (EPS) secreted by the biolm cells acts as a barrier to the diffusion of antibiotic molecules; (ii) the biolm mode of growth leads to altered metabolic activity within the structure together with microscale gradients in nutrient concentration; and (iii) the biolm contains a subpopulation of highly resistant persister cells within the general population. As a consequence of this resistance, biolms associated with implanted medial devices represent a signicant clinical problem and in many cases removal of the device is the only management option. The infecting microorganisms are not always overt pathogens but are often part of the normal microora, thus skin microora such as Staphylococcus epidermidis are frequently found as biolms contaminating implanted devices. Problems have been reported with

a variety of devices, including central venous catheters, urinary catheters, contact lenses, intrauterine contraceptive devices, prosthetic heart valves and orthopaedic implants. Non-medical device-related biolm infections include periodontitis, prostatitis and the chronic P. aeruginosa lung infections associated with cystic brosis. In the absence of effective antibiotic strategies to deal with these infections, phage therapy has been suggested as a possible therapeutic option. However, there are seen to be two main obstacles to the successful infection of biolm cells by bacteriophages. First, the EPS secreted by the adherent cells shields the phage binding sites on the bacterial surface and also acts as a barrier to the penetration of virus into the biolm matrix. Second, the biolm cells within the depths of the structure are oxygen- and nutrient-deprived and consequently are in a state of much reduced metabolic activity. Since bacteriophages require cellular energy for infection to occur, this could potentially militate against a successful outcome. Sutherland and co-workers have shown that some bacteriophages can induce the synthesis of polysaccharide depolymerases in order to degrade the EPS that comprises the bulk of certain biolm matrices [5860]. Enterobacter agglomerans 53b and Serratia marcescens Serr were isolated from biolms occurring within a food factory and both produced signicant quantities of EPS within their biolms. A bacteriophage SF153b could infect and lyse E. agglomerans but not S. marcescens. Similarly, the phage had specic enzymic degradation capability for the EPS of E. agglomerans but not S. marcescens [59]. In single-culture Enterobacter cloacae biolm, a combination of three bacteriophages brought about complete eradication of the cells. However, dual species biolms were not able to be similarly eradicated, suggesting that phages may not be good tools for the treatment of biolm infections. However, most biolm infections are caused by a single species, unlike environmental biolms that are nearly always consortia of multiple species. Very little information is available on the diffusion of bacteriophages through biolm EPS, but other viruses have been shown to degrade mucins to assist their passage toward target cells. Bisaillon et al. [61] showed that reovirus 1 can diffuse through mucus with the aid of a glycosyl hydrolase enzyme. Similarly, baculoviruses possess metalloprotease in their outer coat that can degrade the mucus glycoproteins on the intestinal wall of the insect host. Hanlon et al. [62] observed that P. aeruginosa bacteriophages diffused through alginate gels faster than might be expected and using gel ltration chromatography showed that this was due to degradation of the alginate polymer probably brought about by the presence of an alginate depolymerase enzyme. As this occurred in the absence of bacteria, the enzyme must have been present on the virus itself. Using this phage at a multiplicity of infection of 1000:1, mature 21-day-old biolms were reduced in number by two logs within 24 h. These results suggest that phage enzymes, together with antibiotics, may be

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an alternative control strategy for the management of biolm infections. 7.2. Mycobacterial infections Mycobacteria, in particular M. tuberculosis and Mycobacterium avium, are important causes of chronic disease states that in many cases are highly resistant to antibiotic therapy. Tuberculosis had for centuries been one of the great killer diseases until improvements in socioeconomic circumstances, the introduction of effective vaccination and the advent of antimicrobial therapy saw a remarkable decline in incidence in the early part of the 20th century. Since the mid 1980s, however, the incidence of the disease has risen steadily and, like M. aviumintracellulare, is closely linked with the epidemic of acquired immune deciency syndrome (AIDS). A particular feature both of M. tuberculosis and M. avium infection is the fact that the organisms reside intracellularly within macrophages and monocytes of the host and are able to persist there for extensive periods of time in a latent or dormant phase. This of course poses problems for the potential application of bacteriophages in the treatment of these infections. Sula et al. [63] infected guinea pigs with M. tuberculosis and then treated them with subcutaneous injections of three different bacteriophages twice weekly for 10 weeks. One of these bacteriophages, designated DS-6A, produced an antibacterial effect at least as good as isoniazid. It is unclear, however, whether the phage actually killed the bacteria in their intracellular state. Broxmeyer et al. [64] have shown that the lytic bacteriophage TM4 can be delivered by the transiently infected non-virulent Mycobacterium smegmatis to kill both M. tuberculosis and M. avium residing within macrophages. In this in vitro system, the M. smegmatis vacuoles that harboured the TM4 virus were found to fuse with the M. avium vacuole inside the macrophages. Danelishvili et al. [65] extended this work and studied the ability of M. smegmatis with or without TM4 to kill intracellular M. avium in infected mice. Whilst the phage and bacteria when administered alone produced no therapeutic effect, a signicant reduction in M. avium occurred in the group treated with M. smegmatis containing TM4. Intracellular mycobacteria may therefore be amenable to therapy using lytic phages contained within non-virulent carrier bacteria.

8.1. Bacteriophage lysins Bacteriophage lysins are enzymes produced during the infection process that specically target bacterial peptidoglycan and facilitate exit of viral progeny from the cell. A number of groups have advocated the use of puried lysins as a therapeutic tool rather than infective phage [66]. They have advantages over antibiotics in that they possess the host specicity of phages and so do not adversely affect normal microora; there is less opportunity for resistance to emerge and they kill colonising pathogens on mucosal surfaces [7]. Their major disadvantage is that they are unable to penetrate the outer membrane of Gram-negative cells and so their therapeutic activity is almost entirely directed at Gram-positive infections. Nelson et al. [67] puried the lysin from C1 bacteriophage lytic for the group C streptococcus strain 26RP66 and then used it both to prevent and eliminate colonisation of mice by group A streptococci. They showed that oral administration of the lysin did not affect the indigenous microora but was rapidly lethal to the group A streptococci colonising the mucosal surface of the upper respiratory tract. The preparation was non-irritant and should not induce any mucosal immune response owing to the small quantities applied and its rapidity of action. Increasing concerns over terrorist attacks using biological weapons has led to the exploration of alternative therapies for the most likely candidate agent, anthrax. Inhaled spores can germinate in the lymph nodes surrounding the lungs and secrete toxins into the blood, which is nearly always fatal in untreated patients. Schuch et al. [68] isolated the PlyG lysin from a Bacillus anthracis bacteriophage and showed that this enzyme could kill vegetative cells and germinating spores of the anthrax bacterium both in vitro and in vivo. This concept has been taken a step further by Gaeng et al. [69] who cloned the endolysin genes ply118 and ply511 from bacteriophages of L. monocytogenes into Lactococcus lactis. Listeria monocytogenes is an important food-borne pathogen and is a potential contaminant of foods such as milk and dairy products, therefore the aim of the study was to obtain dairy starter cultures that had biopreservation properties. The genes were expressed under the control of the lactococcal promoter P32 but also required a nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide to be released from the cell. The study showed that the L. lactis cells secreted the enzyme into the surrounding medium and caused rapid lysis of L. monocytogenes contaminants. It may be that approaches such as this might have application in the control of pathogens such as Clostridium difcile in the gut if the normal microora could be supplemented with bacteria coding the appropriate lysin genes. 8.2. Alteration of the host binding prole Bacteriophages can be genetically manipulated to alter their host binding prole. The lamentous phage M13,

8. Novel bacteriophage technologies If bacteriophage therapy is to play a role in infection management in the future, then companies engaged in the manufacture of phage products must be able to protect their intellectual property. Unfortunately, the concept of phage therapy is not new and indeed many of the widely used virulent phages have been obtained from environmental sources. This has led to a number of patents arising that relate to peripheral areas of phage technology, of which a few examples are given below.

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which normally has E. coli as its host, was modied using phage display techniques to bind Helicobacter pylori [70]. Antigen-binding single chain variable fragments from murine hybridomas that produced monoclonal antibodies against H. pylori were expressed as a g3p fusion protein on the M13 phage. The recombinant phage was shown to exhibit a bactericidal effect on a range of H. pylori strains. This technology may have application where a virulent lytic phage cannot be found for a specic host bacterium. 8.3. SASP technology Endospores of Bacillus spp. exhibit profound resistance to heat, radiation and chemicals and this resistance is in part due to the low water content of the spore core. In addition, the spore DNA is protected from damage by saturation with /-type small, acid-soluble spore proteins (SASPs) [7174]. There are three types of SASP, namely , and , which have molecular weights between 5 kDa and 11 kDa. It has been shown that the /-type SASP act as DNAbinding proteins to protect the DNA from damage, whilst the -type SASP is used to supply amino acids for outgrowth [75]. Setlow et al. [76] have shown that a gene encoding an /-type SASP could be inserted into a plasmid under the control of an inducible promoter and this caused a vegetative cell to assume spore-like characteristics. Fairhead [77,78] has employed this technology to develop an antimicrobial system that utilises phage specicity to target the SASP genes into pathogenic bacteria. Bacteriophages are genetically engineered to remove their lysis gene and to replace it with a gene encoding /-type SASP. The phage binds to the specic target bacterium, injects its DNA and all viral genes including SASP are expressed (see Fig. 3). SASP is toxic to the bacterial cell since it binds bacterial DNA irreversibly, halting all cellular activity. The virus does not multiply within bacterium but has been shown to produce rapid killing. The mechanism of action of this system provides limited opportunities for the emergence of resistance and it is being developed as a product targeted at MRSA and C. difcile.

8.4. Engineered prophages Temperate phages induce a state of lysogeny within a bacterial host, where the viral DNA becomes integrated within the host DNA to give rise to what is termed prophage that replicates as the cell replicates. Lysis is an infrequent event. Prophages are commonplace within bacteria but are generally not considered appropriate for therapeutic purposes. However, identifying lytic bacteriophages for certain bacteria can be time consuming because lytic phages for some pathogens are relatively rare and hard to nd. Since prophages are more common in bacteria they are more easily isolated. Some workers have cultivated target bacteria and induced the prophages within them to cause lysis. The vir gene of the bacteriophage progeny is then mutated to give rise to a panel of permanently lytic phages all capable of infecting that specic bacterium [79]. The vir mutants usually contain an alteration in the operator region of the phage DNA that prevents the repressor protein binding to the operator. This alteration leads to derepression of lysogeny, thus transcription and translation of the phage DNA follows and subsequent lysis of the bacterial host. The altered phages have an increased host range compared with the wild-type and the formulated product is targeted in the rst instance at the treatment of nasal carriage of MRSA amongst hospital personnel [80].

9. Summary We have come a long way in our understanding of phage biology since a damning report by Eaton and Bayne Jones in 1934 [81] effectively closed the door on bacteriophage therapy in the West. These viruses have been pivotal in the development of modern molecular biology and in addition those countries that persevered with phage therapy have accumulated many decades of clinical experience. Utilising these two bodies of knowledge, it should be possible to revisit the concept of phage therapy in the West and to have a reasonable expectation of success provided we have an appreciation of the limitations of phage use. Funding: None. Competing interests: None declared. Ethical approval: Not required.

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