1. General Characteristics of Bacteriophages

Hans-Wolfgang Ackermann and Grzegorz Węgrzyn

Bacteriophages are the most numerous biological entities in the biosphere and the largest virus group known. Tailed phages constitute approximately 96% of known phages. Bacteriophage research is an extremely dynamic branch of microbiology. This review starts with the discovery of bacteriophages and covers basic phage techniques, ecology and incidence, aspects of phage physiology and replication, host range, classification, morphology, physico-chemical properties, genomics, and selected practical aspects of phage research. It concludes with a section on electron microscopy for rapid phage identification.

2. The First Step to Bacteriophage Therapy - How to Choose the Correct Phage

Małgorzata Łobocka, Monika S. Hejnowicz, Urszula Gągała, Beata Weber-Dąbrowska, Grzegorz Węgrzyn and Michał Dadlez

Bacteriophages are viruses that can kill bacteria but are harmless to eukaryotic cells. In natural environments they have a dominant role in controlling bacterial populations. Thus, in the era of the emergence and spread of multidrug resistant bacterial pathogens they are more and more often seen as promising antibacterial agents that could be an alternative to antibiotics. Bacteriophages' main advantages as therapeutics are their ability to target bacteria of certain strains or species, without any harmful effect on the rest of the bacterial microflora, as well as their self-limited propagation which is controlled by the availability of a sensitive host. Moreover, bacterial antibiotic resistance is not a barrier for phage infection. Only a limited number of phages from environmental isolates meet the criteria that are expected for therapeutic phages. Here we describe the most important of these criteria and provide a guide for selecting potential therapeutic phages for further studies in animal models. The ability of many phages to remain in a bacterium in the form of a prophage and increase its adaptive potential, as well as to participate in the horizontal gene transfer between bacterial cells, a priori precludes their use in therapy due to safety concerns. Factors that matter in the prediction of the remaining phages' therapeutic efficacy include host range and killing potential, adsorption kinetics and propagation efficiency, stability during storage and under "natural conditions", the ability to penetrate encapsulated cells or biofilms, easiness of purification. The choice or modification of phage propagation host, which cannot be a source of contaminating phages, plasmids and toxins, appears nearly as important as the selection of a proper therapeutic phage.

3. Bacteriophages as Drugs: The Pharmacology of Phage Therapy

Stephen T. Abedon

Pharmacology can be differentiated into two key aspects, pharmacodynamics and pharmacokinetics. Pharmacodynamics describes a drug's impact on the body while pharmacokinetics describes, instead, the body's impact on drugs. Another way of considering these terms is that pharmacodynamics is a description of both the positive and negative consequences of drugs reaching certain densities in the body while pharmacokinetics is concerned with the ability of drugs to reach and sustain those densities. Bacteriophages, or phages, are the viruses of bacteria. Here I consider the pharmacology of phages as drugs or - as they are applied to treat bacterial infections (phage therapy) - at least as drug-like entities. I suggest that phages, contrasting other narrow-spectrum but chemical antibacterials, possess a unique combination of properties including ease of discovery and laboratory characterization, low toxicity and minimal side effects, and the potential to amplify their numbers in situ. The result of these somewhat un-chemotherapeutic-like aspects can mean that detailed exploration especially of phage therapy pharmacokinetics in many instances may be somewhat superfluous. Nonetheless, an improved pharmacological understanding of phage therapy should allow for more informed development, as well as rational post hoc debugging, of phage therapy experiments and improved phage therapy design.

4. Fighting Bacteriophage Infection: Mechanisms of Bacterial Resistance

Anneleen Cornelissen, Rob Lavigne and Sylvain Moineau

In man-made and natural environments, there is a continuous ongoing interaction between phages and their bacterial hosts, a co-evolutionary arms race between two competing organisms which contributes enormously to their diversity. During continuous cycles of co-evolution, phage-resistant bacterial hosts emerge aiming at preservation of their bacterial lineages. For every step in the phage infection cycle, bacteria have evolved various defense mechanisms, passive or active, to evade phage propagation and subsequent spreading of phage progeny in the surrounding environment. However, when facing this selective pressure imposed by the host, phages have developed different strategies to subvert these defense systems in order to thrive in these new bacterial populations. Knowledge of these phage-host dynamics represents a vital tool for phage therapeutic purposes in which the emergence of phage-resistant bacterial pathogen forms a notable disadvantage. In contrast, in the fermentation industries, bacteriophages themselves pose a contamination problem, which can be relieved by selection of phage-insensitive bacterial strains.

5. Non-bactericidal Effects of Phages in Mammals

Krystyna Dąbrowska, Ryszard Międzybrodzki, Paulina Miernikiewicz, Grzegorz Figura and Andrzej Górski

Bacteriophages, although unable to propagate in eukaryotic cells, may induce physiological effects in mammalian organisms. Phage impact on the immune system as well as phage interactions with its elements may decide on the final outcome of phage therapy, thus being of a great practical importance for medical applications of phages. The most spectacular but also expected effect of phages in living organisms is the induction of anti-phage antibodies. These have been showed to play a role in phage clearance. However, phages have been shown to be able to modify ROS and cytokine production in mammalian immunological cells. Phage interaction with mammalian cells can be, at least partially, mediated by direct adhesion of bacterial viruses to the cells. Phages are abundant parasites of symbiotic or pathogenic bacteria in animals and humans. Consequently, mammals have become an 'environment' for phages. This environment is a multi-factor system able to induce strong pressure. Phages are complex structures, potentially able to evolve new means of interacting with their environment. Apart from natural phages, engineered phage particles are gaining an important position in biotechnology and medicine. They can be applied as carriers for vaccines or other biologically active agents. These applications, together with phage therapy of bacterial infections, induce constantly growing interest in interactions of phages with mammalian systems.

6. Overview of Therapeutic Applications of Bacteriophages

David Kelly, Olivia McAuliffe, R. Paul Ross, Jim O'Mahony and Aidan Coffey

This chapter will give an overview of the different applications of bacteriophages (phages). These include the use of whole virulent phages as antibacterials, recent advances in the development and applications of genetically modified phages, the use of phages as delivery vehicles and vaccines, and recent developments in the study and use of purified endolysins. Generally, it is clear that phages have a considerable array of potential applications as therapeutics in the modern medical and veterinary fields. In particular, the continued antibiotic resistance problem in clinically relevant micro-organisms calls for the exploitation of cheap, natural, readily-available, safe and efficient therapeutic agents. Phages exhibit characteristics which satisfy all of these criteria and their reintroduction as medical treatment options is worthy of strong consideration.

7. Considerations for Using Bacteriophages in Plant Pathosystems

Jeffrey B. Jones, Aleksa Obradovic and Botond Balogh

Phages have the potential for controlling plant pathogens in the rhizosphere or phyllosphere. Success of phage in disease control requires that high populations of both phage and bacterium exist in order to initiate a chain reaction of bacterial lysis. Various factors exist that can hinder success of disease control. Physical factors in natural environments such as the presence of biofilms that trap bacteriophages, low soil pH which inactivates phages, low rates of diffusion of phages in soil that prevent contact with target bacteria, and inactivation of phages upon exposure to UV, all impact successful use of phages. Other considerations relate to the bacterial strains which exist in nature. The bacterial species may have a low or high degree of variation in sensitivity to bacteriophages. Therefore, phage selection for field use requires careful monitoring of strains in the field be done due to the potential for strain variation in the field and the likelihood for development of bacterial strains with resistance to the deployed bacteriophages. Application timing has also been shown to be an important factor in improving efficacy of bacteriophages. For instance, ultraviolet light is deleterious to bacteriophages and upon exposure phage populations plummet; therefore, evening applications of bacteriophages result in persistence of phages on leaf surfaces for longer periods of time and may result in improved disease control. Extending the period of time phages persist in the phyllosphere has been a major hurdle. Formulations have been identified which improve the persistence of bacteriophages on leaf surfaces; however, there is a need to identify superior formulations that extend the life on leaf surfaces from hours to days. Another strategy for maintaining high populations of phages has been to use non-pathogenic bacterial strains that are sensitive to the phage(s) or a closely related organism that does not cause disease on the plant host. Finally, bacteriophages may have value as part of an integrated management strategy.

8. Bacteriophage Therapy in Animal Production

William E. Huff and Geraldine R. Huff

Concerns over the consequences of bacterial resistance to antibiotics with the use of antibiotics in animal production have led to an increase in research on alternatives to antibiotics. Bacteriophages kill bacteria, are natural, safe, plentiful, self replicating, self limiting, can be used to specifically target pathogens without disruption of commensal bacteria, and have diverse biological properties. These properties make bacteriophages an attractive alternative to antibiotics, especially applicable for the control of antibiotic resistant bacteria. The efficacy of bacteriophages to prevent and treat animal diseases has been shown in almost all production animals in both laboratory and commercial field studies, without any adverse affects in the animals. Although the potential of bacteriophage to control significant diseases in animal production has been demonstrated, bacteriophage therapeutics do not represent a replacement of antibiotics. There are some applications in animal production systems where bacteriophage therapeutics have an advantage over the use of antibiotics and some applications where bacteriophage therapeutics are at a disadvantage over the use of antibiotics. In addition, the effectiveness of antibiotics and bacteriophage therapy can be enhanced when combined to treat animal diseases. The objectives of this chapter are to review the literature documenting the efficacy of bacteriophages to control diseases in animal production, to discuss the advantages and disadvantages of bacteriophage therapy, and to describe possible applications for the use of bacteriophages to control bacterial diseases in commercial poultry, swine, cattle, and aquaculture systems.

9. The Use of Phages as Biocontrol Agents in Foods

Jan Borysowski and Andrzej Górski

Bacteriophages have several features which make them novel antibacterial agents with which to prevent bacterial foodborne infections. Phages can be used to control the growth of bacteria both in food products and on food contact surfaces. In recent years, many studies have shown relatively high efficacy of phages against several major foodborne pathogens, especially Listeria monocytogenes, Salmonella enterica, Escherichia coli including E. coli O157:H7, Campylobacter jejuni, and Staphylococcus aureus in a variety of foods; phages were also successfully used against Yersinia enterocolitica, Shigella spp., Bacillus cereus, and Cronobacter spp. In addition, some attempts were made to use phages to eliminate food spoilage bacteria. In most studies, the use of phages resulted in significant, log-fold reductions in the bacterial counts in foods; such reductions are known to substantially decrease a risk of foodborne infections. Main factors that determine the efficacy of bacteriophages as biocontrol agents include phage particles density in/on a food product, a level of bacterial contamination, the development of bacterial resistance to phages, and phage stability in different foods. Recent clearance by FDA of four bacteriophage preparations for food applications shows that bacteriophages are gradually gaining acceptance as a means of prevention of foodborne infections.

10. Phage Therapy: Experiments Using Animal Infection Models

Shigenobu Matsuzaki, Jumpei Uchiyama, Iyo Takemura-Uchiyama and Masanori Daibata

Phage therapy is a method of controlling bacterial infections by bacteriophage (phage) administration. Many animal experiments have been performed to demonstrate the efficacy and safety of phage therapy. Bacterial species that are currently prevalent in clinical settings have been selected as the targets of experimental phage therapy. These include Gram-positive bacteria such as Staphylococcus aureus including methicillin-resistant strains (MRSA) and Enterococcus species including vancomycin-resistant strains (VRE), as well as Gram-negative bacteria such as Pseudomonas aeruginosa (and its relatives), Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumanii. The infections selected have included meningitis, lung infections, wound infections, urinary tract infections, gastrointestinal infections, and septicemia, all of which are clinically relevant. In most animal experiments, phage therapy has been demonstrated to be very effective and safe. Furthermore, phage therapy has been shown to be effective in immunosuppressed and diabetic animals, as well as in a murine model of chronic infection. Methods to overcome the problem of anti-phage antibodies and of phage therapy against intracellular bacteria such as Mycobacterium species have also been demonstrated. Moreover, genetically modified phages, which can kill the host bacteria without lysing them, have also been developed, and their efficacy has been demonstrated. This accumulated evidence encourages us to develop phage therapy systems for the future treatment of human infections.

11. Clinical Phage Therapy

Elizabeth Kutter, Jan Borysowski, Ryszard Międzybrodzki, Andrzej Górski, Beata Weber-Dąbrowska, Mzia Kutateladze, Zemphira Alavidze, Marina Goderdzishvili and Revaz Adamia

Since the first therapeutic use of phages in humans in 1919, a large number of studies have been conducted to evaluate the safety and the efficacy of phage therapy in a wide range of bacterial infections, and phage therapy has become a well-accepted part of clinical practice in some parts of the world. Elsewhere, interest in phage therapy has been growing strongly in parallel with the emerging threats of antibiotic resistance worldwide and the paucity of new approaches, but progress has been hampered by the lack of the rigorous double-blind clinical trials now widely required for governmental approval and the challenges in getting funding for such studies. Earlier therapeutic phage applications are important for further development of clinical phage therapy in multiple ways. First, their results strongly suggest the safety of bacteriophage preparations; no serious side effects have been reported despite their use by hundreds of thousands of people, suggesting that any such effects are almost certainly rare and/or subtle. Secondly, they may help to select specific diseases and approaches to be best targeted by future more tightly controlled trials of phage therapy. Thirdly, more in-depth exploration of the strongest earlier work and of recent advances in the field should help spur private, governmental and academic investment in further research. The main goal of this chapter is to discuss in detail the recent controlled trials of bacteriophage preparations along with the clinical experiences of the two major centers of phage therapy, the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wrocław, Poland, and the George Eliava Institute of Bacteriophages, Microbiology and Virology, Tbilisi, Georgia. The positive results of hitherto conducted studies, as well as dramatic increase in the prevalence of antibiotic-resistant bacteria, warrant concerted further explorations of clinical phage therapy as one element in our arsenal.

12. Reintroducing Phage Therapy in Modern Medicine: The Regulatory and Intellectual Property Hurdles

Daniel De Vos, Gilbert Verbeken, Carl Ceulemans, Isabelle Huys and Jean-Paul Pirnay

Antibiotic resistance is a life-threatening problem worldwide and the industrial pipeline is dry. Other therapeutic options are needed and one of them is 'phage therapy'. Bacteriophages, phage in short, have proven to be effective in combating (multidrug-resistant) bacterial infections. However, legal obstacles and intellectual property rights are impeding the implementation of phage therapy in modern medicine and triggering ethical discussions. Worldwide, medicinal product regulations are directed towards standardized marketing authorization for 'classical' medicinal products. But phage are of a different nature than antibiotics on which most of our current regulation is based. Phage therapy is not covered by a specific regulatory pathway. Exceptions defined under the medicinal products legislation do not include the idea of phage therapy. Another hurdle is the Intellectual Property issue. Owning patents is essential in our current industrial economic model. But natural phages are evolving biological lifelike entities and thus difficult to cover by patents. In the future the adapted legal framework should allow the coexistence of a "sur-mesure" pathway beside a "prêt-à-porter" road. Taking into account the sustainability concept, all relevant safety measures and quality production controls, the "sur-mesure" pathway should enable the use of the most fruitful and efficiency based phage therapeutic approach at regional or hospital level.

13. The Use of Bacteriophages and Bacteriophage-derived Enzymes for Clinically Relevant Biofilm Control

Sanna Sillankorva and Joana Azeredo

Biofilm formation occurs spontaneously on both inert and living systems and is an important bacterial survival strategy. Biofilms are often associated with several chronic and acute infections such as wound infections, vaginitis, upper respiratory tract infections, otitis media, and endocarditis among others. There is an inherent tolerance of cells embedded in biofilms towards antibiotics and therefore, alternative agents, such as bacteriophages have been the focus of interest. This is an overview of the phage-biofilm interaction research carried out, starting with a general introduction to biofilm structure and composition, and to clinically relevant biofilms and their inherent tolerance to antimicrobials. Then, the use of phages and phage derived enzymes as alternative biofilm control agents will be discussed in detail based on the results from a number of in vitro experiments found in literature. Although focus is almost exclusively given to the results of biofilm control and biofilm prevention experiments using phages and phage-derived enzymes, other relevant topics, such as the diffusion of phages through the matrix, and the effect of prophages, will also be addressed.

14. Using What Phage Have Evolved to Kill Pathogenic Bacteria

Vincent A. Fischetti

Bacteriophage (or phage) endolysins (or lysins) are highly evolved enzymes produced to cleave essential bonds in the bacterial cell wall peptidoglycan for phage progeny release. Small quantities of purified recombinant lysin added externally to gram-positive bacteria cause immediate lysis resulting in log-fold death of the target bacterium. Lysins have now been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and in infected tissues. The advantages over antibiotics are their specificity for the pathogen even antibiotic resistant organisms without disturbing the normal flora, the low chance of bacterial resistance, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Lysins therefore, may be a much-needed anti-infective (or enzybiotic) in an age of mounting antibiotic resistance.

15. Genetically-engineered Phage as Antimicrobials and Biodetectors

Salim Manoharadas and Udo Bläsi

With the advent of antibiotics the use of bacteriophage as antimicrobial agents has been abandoned in the western world. However, the increasing prevalence of multi-drug resistant bacterial pathogens has resulted in a resurgence of research efforts to use phage as antimicrobials. A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g. endotoxins of Gram-negative bacteria, which mediate the general pathological aspects of septicemia. In the last decade several strategies based on genetically engineered lysis-deficient phage have been devised with the aim to avoid disintegration of the cell envelope but to kill the bacterial target. These studies indicated that killing-proficient but lysis-defective recombinant phage can be exploited as efficient antimicrobials with reduced side effects. Moreover, genetically engineered phage can be used to augment the antimicrobial efficacy of antibiotics and to reduce bacterial biofilms. Apart from these potential medical applications, modified phages have been used to detect bacterial pathogens in foodstuff. Here, we provide a review of these studies and briefly discuss the prospects of genetically modified phage in medicine and industry.

16. Engineered Filamentous Bacteriophages as Targeted Anti-bacterial Drug-carrying Nanomedicines

Lilach Vaks and Itai Benhar

The increasing development of bacterial resistance to traditional antibiotics forces the scientists to develop new antimicrobial approaches. In traditional as well as newly developed antibiotics, the drug itself provides the target specificity, thus excluding potent but non-selective drugs from possible use. The conjugation of a toxic drug to a targeted carrier replaces the drug selectivity by the targeting moiety of the carrier thus maximizing the drug bio-availability to diseased tissues and minimizing the healthy tissues exposure. Bacteriophages (phages) possess unique characteristics such as modular nanometric structure, high specificity to host bacteria, an ability to display foreign proteins on the phage surface and varying levels of tolerance to chemical modification which suggest they may be perfect carriers for targeting and eradication of bacterial pathogens. The natural ability of host-specific phages to infect and lyse their host bacteria in animal disease models was already demonstrated in early 1940s. However, major challenges such as limited host specificity, uncontrolled replication and high immunogenicity remained mostly unaddressed. The application of phage carriers loaded with non-selective antibacterial drugs provides for drug accumulation at high concentration vicinal to the target pathogen and its effective killing with minimal effect on the organism infected by the pathogen. The targeting moiety is provided by either natural host-specific recognition or by genetic modification of the phage to display pathogen-specific peptide or antibody on phage surface. Such approaches were demonstrated mostly with filamentous phages, but also with tailed phages. The drug conjugation affects the phage in vivo properties and decreases its immunogenicity. In conclusion, the unique natural properties of the phage carrier and its universal compatibility for drug conjugation and for display of target-specific proteins and peptides give the phage nanoparticle an exclusive advantage among the presently existing anti bacterial drug-delivery systems.