Translating phage therapy into the clinic: Recent accomplishments but continuing challenges.

Phage therapy is a medical form of biological control of bacterial infections, one that uses naturally occurring viruses, called bacteriophages or phages, as antibacterial agents. Pioneered over 100 years ago, phage therapy nonetheless is currently experiencing a resurgence in interest, with growing numbers of clinical case studies being published. This renewed enthusiasm is due in large part to phage therapy holding promise for providing safe and effective cures for bacterial infections that traditional antibiotics acting alone have been unable to clear. This Essay introduces basic phage biology, provides an outline of the long history of phage therapy, highlights some advantages of using phages as antibacterial agents, and provides an overview of recent phage therapy clinical successes. Although phage therapy has clear clinical potential, it faces biological, regulatory, and economic challenges to its further implementation and more mainstream acceptance.

How Simple Maths Can Inform Our Basic Understanding of Phage Therapy.

Phage therapy is the application of bacterial viruses to control and, ideally, to eliminate problematic bacteria from patients. Usually employed are so-called strictly lytic phages, which upon adsorption of a bacterium should give rise to both bacterial death and bacterial lysis. This killing occurs with single-hit kinetics, resulting in relatively simple ways to mathematically model organismal-level, phage-bacterium interactions. Reviewed here are processes of phage therapy as viewed from these simpler mathematical perspectives, starting with phage dosing, continuing through phage adsorption of bacteria, and then considering the potential for phage numbers to be enhanced through in situ phage population growth. Overall, I suggest that a basic working knowledge of the underlying "simple maths" of phage therapy can be helpful toward making dosing decisions and predicting certain outcomes. This especially is during controlled in vitro experimentation but is relevant to thinking about in vivo applications as well.

Phage Therapy: The Pharmacology of Antibacterial Viruses.

Pharmacology can be differentiated into two key aspects, pharmacodynamics and pharmacokinetics. Pharmacodynamics describes a drug's impact on the body while pharmacokinetics describes the body's impact on a drug. Another way of understanding these terms is that pharmacodynamics is a description of both the positive and negative consequences of drugs attaining certain concentrations in the body while pharmacokinetics is concerned with our ability to reach and then sustain those concentrations. Unlike the drugs for which these concepts were developed, including antibiotics, the bacteriophages (or 'phages') that we consider here are not chemotherapeutics but instead are the viruses of bacteria. Here we review the pharmacology of these viruses, particularly as they can be employed to combat bacterial infections (phage therapy). Overall, an improved pharmacological understanding of phage therapy should allow for more informed development of phages as antibacterial 'drugs', allow for more rational post hoc debugging of phage therapy experiments, and encourage improved design of phage therapy protocols. Contrasting with antibiotics, however, phages as viruses impact individual bacterial cells as single virions rather than as swarms of molecules, and while they are killing bacteria, bacteriophages also can amplify phage numbers, in situ. Explorations of phage therapy pharmacology consequently can often be informed as well by basic principles of the ecological interactions between phages and bacteria as by study of the pharmacology of drugs. Bacteriophages in phage therapy thus can display somewhat unique as well as more traditional pharmacological aspects.

Archaeal viruses, not archaeal phages: an archaeological dig.

Viruses infect members of domains Bacteria, Eukarya, and Archaea. While those infecting domain Eukarya are nearly universally described as "Viruses", those of domain Bacteria, to a substantial extent, instead are called "Bacteriophages," or "Phages." Should the viruses of domain Archaea therefore be dubbed "Archaeal phages," "Archaeal viruses," or some other construct? Here we provide documentation of published, general descriptors of the viruses of domain Archaea. Though at first the term "Phage" or equivalent was used almost exclusively in the archaeal virus literature, there has been a nearly 30-year trend away from this usage, with some persistence of "Phage" to describe "Head-and-tail" archaeal viruses, "Halophage" to describe viruses of halophilic Archaea, use of "Prophage" rather than "Provirus," and so forth. We speculate on the root of the early 1980's transition from "Phage" to "Virus" to describe these infectious agents, consider the timing of introduction of "Archaeal virus" (which can be viewed as analogous to "Bacterial virus"), identify numerous proposed alternatives to "Archaeal virus," and also provide discussion of the general merits of the term, "Phage." Altogether we identify in excess of one dozen variations on how the viruses of domain Archaea are described, and document the timing of both their introduction and use.

Ecology and Evolutionary Biology of Hindering Phage Therapy: The Phage Tolerance vs. Phage Resistance of Bacterial Biofilms.

As with antibiotics, we can differentiate various acquired mechanisms of bacteria-mediated inhibition of the action of bacterial viruses (phages or bacteriophages) into ones of tolerance vs. resistance. These also, respectively, may be distinguished as physiological insensitivities (or protections) vs. resistance mutations, phenotypic resistance vs. genotypic resistance, temporary vs. more permanent mechanisms, and ecologically vs. also near-term evolutionarily motivated functions. These phenomena can result from multiple distinct molecular mechanisms, many of which for bacterial tolerance of phages are associated with bacterial biofilms (as is also the case for the bacterial tolerance of antibiotics). The resulting inhibitions are relevant from an applied perspective because of their potential to thwart phage-based treatments of bacterial infections, i.e., phage therapies, as well as their potential to interfere more generally with approaches to the phage-based biological control of bacterial biofilms. In other words, given the generally low toxicity of properly chosen therapeutic phages, it is a combination of phage tolerance and phage resistance, as displayed by targeted bacteria, that seems to represent the greatest impediments to phage therapy's success. Here I explore general concepts of bacterial tolerance of vs. bacterial resistance to phages, particularly as they may be considered in association with bacterial biofilms.

Automating Predictive Phage Therapy Pharmacology.

Viruses that infect as well as often kill bacteria are called bacteriophages, or phages. Because of their ability to act bactericidally, phages increasingly are being employed clinically as antibacterial agents, an infection-fighting strategy that has been in practice now for over one hundred years. As with antibacterial agents generally, the development as well as practice of this phage therapy can be aided via the application of various quantitative frameworks. Therefore, reviewed here are considerations of phage multiplicity of infection, bacterial likelihood of becoming adsorbed as a function of phage titers, bacterial susceptibility to phages also as a function of phage titers, and the use of Poisson distributions to predict phage impacts on bacteria. Considered in addition is the use of simulations that can take into account both phage and bacterial replication. These various approaches can be automated, i.e., by employing a number of online-available apps provided by the author, the use of which this review emphasizes. In short, the practice of phage therapy can be aided by various mathematical approaches whose implementation can be eased via online automation.

Schlesinger Nailed It! Assessing a Key Primary Pharmacodynamic Property of Phages for Phage Therapy: Virion Encounter Rates with Motionless Bacterial Targets.

Bacteriophages (phages) are viruses of bacteria and have been used as antibacterial agents now for over one-hundred years. The primary pharmacodynamics of therapeutic phages can be summed up as follows: phages at a certain concentration can reach bacteria at a certain rate, attach to bacteria that display appropriate receptors on their surfaces, infect, and (ideally) kill those now-adsorbed bacteria. Here, I consider the rate at which phages reach bacteria, during what can be dubbed as an 'extracellular search'. This search is driven by diffusion and can be described by what is known as the phage adsorption rate constant. That constant in turn is thought to be derivable from knowledge of bacterial size, virion diffusion rates, and the likelihood of phage adsorption given this diffusion-driven encounter with a bacterium. Here, I consider only the role of bacterial size in encounter rates. In 1932, Schlesinger hypothesized that bacterial size can be described as a function of cell radius (R, or R1), as based on the non-phage-based theorizing of Smoluchowski (1917). The surface area of a cell-what is actually encountered-varies however instead as a function R2. Here, I both provide and review evidence indicating that Schlesinger's assertion seems to have been correct.

Phage therapy pharmacology phage cocktails.

Phage therapy is the clinical or veterinary application of bacterial viruses (bacteriophages) as antibacterial "drugs." More generally, phages can be used as biocontrol agents against plant as well as foodborne pathogens. In this chapter, we consider the therapeutic use of phage cocktails, which is the combining of two or more phage types to produce more pharmacologically diverse formulations. The primary motivation for the use of cocktails is their broader spectra of activity in comparison to individual phage isolates: they can impact either more bacterial types or achieve effectiveness under a greater diversity of conditions. The combining of phages can also facilitate better targeting of multiple strains making up individual bacterial species or covering multiple species that might be responsible for similar disease states, in general providing, relative to individual phage isolates, a greater potential for presumptive or empirical treatment. Contrasting the use of phage banks, or even phage isolation against specific etiologies that have been obtained directly from patients under treatment, here we consider the utility as well as potential shortcomings associated with the use of phage cocktails as therapeutic antibacterial agents.

Pathways to Phage Therapy Enlightenment, or Why I Have Become a Scientific Curmudgeon.

Over the past decade I, with collaborators, have authored a number of publications outlining what in the first of these I described as "Phage therapy best practices"-phage therapy being the use of bacterial viruses (bacteriophages) to treat bacterial infections, such as clinically. More generally, this is phage-mediated biocontrol of bacteria, including of bacteria that can contaminate foods. For the sake of increasing accessibility, here I gather some of these suggestions, along with some frustrations, into a single place, while first providing by way of explanation where they, and I, come from scientifically. Although in my opinion phage therapy and phage-mediated biocontrol are both sound approaches toward combating unwanted bacteria, I feel at the same time that the practice of especially phage therapy research could be improved. I supply also, as supplemental material, a list of ∼100 English language 2000-and-later publications providing primary descriptions of phage application to humans.

Further Considerations on How to Improve Phage Therapy Experimentation, Practice, and Reporting: Pharmacodynamics Perspectives.

Phage therapy uses bacterial viruses (bacteriophages) to infect and kill targeted pathogens. Approximately one decade ago, I started publishing on how possibly to improve upon phage therapy experimentation, practice, and reporting. Here, I gather and expand upon some of those suggestions. The issues emphasized are (1) that using ratios of antibacterial agents to bacteria is not how dosing is accomplished in the real world, (2) that it can be helpful to not ignore Poisson distributions as a means of either anticipating or characterizing phage therapy success, and (3) how to calculate a concept of 'inundative phage densities.' Together, these are issues of phage therapy pharmacodynamics, meaning they are ways of thinking about the potential for phage therapy treatments to be efficacious mostly independent of the details of delivery of phages to targeted bacteria. Much emphasis is placed on working with Poisson distributions to better align phage therapy with other antimicrobial treatments.

Virus-Like Particle: Evolving Meanings in Different Disciplines.

Virus-like particle (VLP) is a term that has been in use for about 80 years. Usually, VLP has meant a particle that is like a virus, generally by appearance, but without either proven or actual virus functionality. Initially VLP referred to particles seen in electron microscope images of tissues. More recently, VLP has come to mean other things to other researchers. A key divergence has been use of VLP in association with vaccine and biotechnology applications versus use of VLP in enumeration of viruses in environmental samples. To these viral ecologists, a VLP is a particle that is virus sized, has nucleic acid, and could be a functional virus. But to vaccine developers and biotechnology researchers a VLP instead is a viral structure that intentionally lacks a viral genome. In this study, we look at the history of use of VLP, following changes in meaning as the technology to study VLPs changed.

Treating Bacterial Infections with Bacteriophage-Based Enzybiotics: In Vitro, In Vivo and Clinical Application.

Over the past few decades, we have witnessed a surge around the world in the emergence of antibiotic-resistant bacteria. This global health threat arose mainly due to the overuse and misuse of antibiotics as well as a relative lack of new drug classes in development pipelines. Innovative antibacterial therapeutics and strategies are, therefore, in grave need. For the last twenty years, antimicrobial enzymes encoded by bacteriophages, viruses that can lyse and kill bacteria, have gained tremendous interest. There are two classes of these phage-derived enzymes, referred to also as enzybiotics: peptidoglycan hydrolases (lysins), which degrade the bacterial peptidoglycan layer, and polysaccharide depolymerases, which target extracellular or surface polysaccharides, i.e., bacterial capsules, slime layers, biofilm matrix, or lipopolysaccharides. Their features include distinctive modes of action, high efficiency, pathogen specificity, diversity in structure and activity, low possibility of bacterial resistance development, and no observed cross-resistance with currently used antibiotics. Additionally, and unlike antibiotics, enzybiotics can target metabolically inactive persister cells. These phage-derived enzymes have been tested in various animal models to combat both Gram-positive and Gram-negative bacteria, and in recent years peptidoglycan hydrolases have entered clinical trials. Here, we review the testing and clinical use of these enzymes.

Phage Therapy in the 21st Century: Is There Modern, Clinical Evidence of Phage-Mediated Efficacy?

Many bacteriophages are obligate killers of bacteria. That this property could be medically useful was first recognized over one hundred years ago, with 2021 being the 100-year anniversary of the first clinical phage therapy publication. Here we consider modern use of phages in clinical settings. Our aim is to answer one question: do phages serve as effective anti-bacterial infection agents when used clinically? An important emphasis of our analyses is on whether phage therapy-associated anti-bacterial infection efficacy can be reasonably distinguished from that associated with often coadministered antibiotics. We find that about half of 70 human phage treatment reports-published in English thus far in the 2000s-are suggestive of phage-mediated anti-bacterial infection efficacy. Two of these are randomized, double-blinded, infection-treatment studies while 14 of those studies, in our opinion, provide superior evidence of a phage role in observed treatment successes. Roughly three-quarters of these potentially phage-mediated outcomes are based on microbiological as well as clinical results, with the rest based on clinical success. Since many of these phage treatments are of infections for which antibiotic therapy had not been successful, their collective effectiveness is suggestive of a valid utility in employing phages to treat otherwise difficult-to-cure bacterial infections.

Phage Cocktail Development for Bacteriophage Therapy: Toward Improving Spectrum of Activity Breadth and Depth.

Phage therapy is the use of bacterial viruses as antibacterial agents. A primary consideration for commercial development of phages for phage therapy is the number of different bacterial strains that are successfully targeted, as this defines the breadth of a phage cocktail's spectrum of activity. Alternatively, phage cocktails may be used to reduce the potential for bacteria to evolve phage resistance. This, as we consider here, is in part a function of a cocktail's 'depth' of activity. Improved cocktail depth is achieved through inclusion of at least two phages able to infect a single bacterial strain, especially two phages against which bacterial mutation to cross resistance is relatively rare. Here, we consider the breadth of activity of phage cocktails while taking both depth of activity and bacterial mutation to cross resistance into account. This is done by building on familiar algorithms normally used for determination solely of phage cocktail breadth of activity. We show in particular how phage cocktails for phage therapy may be rationally designed toward enhancing the number of bacteria impacted while also reducing the potential for a subset of those bacteria to evolve phage resistance, all as based on previously determined phage properties.

Improving Phage-Biofilm In Vitro Experimentation.

Bacteriophages or phages, the viruses of bacteria, are abundant components of most ecosystems, including those where bacteria predominantly occupy biofilm niches. Understanding the phage impact on bacterial biofilms therefore can be crucial toward understanding both phage and bacterial ecology. Here, we take a critical look at the study of bacteriophage interactions with bacterial biofilms as carried out in vitro, since these studies serve as bases of our ecological and therapeutic understanding of phage impacts on biofilms. We suggest that phage-biofilm in vitro experiments often may be improved in terms of both design and interpretation. Specific issues discussed include (a) not distinguishing control of new biofilm growth from removal of existing biofilm, (b) inadequate descriptions of phage titers, (c) artificially small overlying fluid volumes, (d) limited explorations of treatment dosing and duration, (e) only end-point rather than kinetic analyses, (f) importance of distinguishing phage enzymatic from phage bacteriolytic anti-biofilm activities, (g) limitations of biofilm biomass determinations, (h) free-phage interference with viable-count determinations, and (i) importance of experimental conditions. Toward bettering understanding of the ecology of bacteriophage-biofilm interactions, and of phage-mediated biofilm disruption, we discuss here these various issues as well as provide tips toward improving experiments and their reporting.

Friends or Foes? Rapid Determination of Dissimilar Colistin and Ciprofloxacin Antagonism of Pseudomonas aeruginosa Phages.

Phage therapy is a century-old technique employing viruses (phages) to treat bacterial infections, and in the clinic it is often used in combination with antibiotics. Antibiotics, however, interfere with critical bacterial metabolic activities that can be required by phages. Explicit testing of antibiotic antagonism of phage infection activities, though, is not a common feature of phage therapy studies. Here we use optical density-based 'lysis-profile' assays to assess the impact of two antibiotics, colistin and ciprofloxacin, on the bactericidal, bacteriolytic, and new-virion-production activities of three Pseudomonas aeruginosa phages. Though phages and antibiotics in combination are more potent in killing P. aeruginosa than either acting alone, colistin nevertheless substantially interferes with phage bacteriolytic and virion-production activities even at its minimum inhibitory concentration (1× MIC). Ciprofloxacin, by contrast, has little anti-phage impact at 1× or 3× MIC. We corroborate these results with more traditional measures, particularly colony-forming units, plaque-forming units, and one-step growth experiments. Our results suggest that ciprofloxacin could be useful as a concurrent phage therapy co-treatment especially when phage replication is required for treatment success. Lysis-profile assays also appear to be useful, fast, and high-throughput means of assessing antibiotic antagonism of phage infection activities.

Bacteriophage host range and bacterial resistance.

Host range describes the breadth of organisms a parasite is capable of infecting, with limits on host range stemming from parasite, host, or environmental characteristics. Parasites can adapt to overcome host or environmental limitations, while hosts can adapt to control the negative impact of parasites. We consider these adaptations as they occur among bacteriophages (phages) and their bacterial hosts, since they are significant to phage use as antibacterials (phage therapy) or to protection of industrial ferments from phage attack. Initially, we address how phage host range can (and should) be defined plus summarize claims of host ranges spanning multiple bacterial genera. Subsequently, we review bacterial mechanisms of phage resistance. These include adsorption resistance, which results in reduced interaction between phage and bacterium; what we describe as "restriction," where bacteria live but phages die; and abortive infections, where both phage and bacterium die. Adsorption resistance includes loss of phage receptor molecules on hosts as well as physical barriers hiding receptor molecules (e.g., capsules). Restriction mechanisms include phage-genome uptake blocks, superinfection immunity, restriction modification, and CRISPR, all of which function postphage adsorption but prior to terminal phage takeover of host metabolism. Standard laboratory selection methods, involving exposure of planktonic bacteria to high phage densities, tend to directly select for these prehost-takeover resistance mechanisms. Alternatively, resistance mechanisms that do not prevent bacterium death are less readily artificially selected. Contrasting especially bacteria mutation to adsorption resistance, these latter mechanisms likely are an underappreciated avenue of bacterial resistance to phage attack.

Phage evolution and ecology.

Bacteriophages (phages) are the viruses of bacteria and the study of phage biology can be differentiated, roughly, into molecular, environmental, evolutionary, ecological, and applied aspects. While for much of the past fifty-plus years molecular and then applied aspects have dominated the field, more recently environmental concerns, especially the phage impact on biogeochemical cycles, have driven an increase in the appreciation of phage ecology. Over approximately the same time frame, decreasing sequencing costs have combined with phage molecular characterization to give rise to an inescapable consideration of phage comparative genomics. That, along with environmental metagenomics, has stimulated, especially among molecular biologists, a more general interest in phage evolutionary biology. However, while reviews of phage ecology have become exceedingly common, overviews of phage evolutionary biology are comparatively rare, and broad considerations of phage evolutionary biology drawn from an ecological perspective rarer still. In this chapter I jump into this latter void, providing an overview of phage evolutionary biology as viewed from the perspective of phage-environment interactions, that is, from the perspective of phage ecology. This I do over five sections constituting (1) an introduction to phages and how, phenotypically, they can be differentiated into three basics types that correlate, more or less, with genomic size, that is, tailed (generally larger genomes), lipid-containing (medium-sized genomes), and single-stranded (small genomes); (2) a brief introduction to phage ecology as considered particularly from a classical ecological perspective; (3) an extended introduction to evolutionary biology as viewed from a phage and phage-ecological standpoint; (4) phage evolutionary ecology, that is, consideration of phage adaptations from the vantage of why, in terms of phage fitness, those adaptations may have evolved; and (5) phage evolutionary biology, including evolutionary ecology, as viewed from the perspective of phage genomics.

Bacteriophage plaques: theory and analysis.

Laboratory characterization of bacteriophage growth traditionally is done either in broth cultures or in semisolid agar media. These two environments may be distinguished in terms of their spatial structure, i.e., the degree to which they limit diffusion, motility, and environmental mixing. Well-mixed broth, for example, represents the microbiological ideal of a non-spatially structured environment. Agar, by contrast, imposes significant limitations on phage and bacterial movement and therefore gives rise to spatial structure. The study of phage growth within spatially structured environments, such as that seen during phage plaque formation, is important for three reasons. First, a large fraction of environmental bacteria live within spatially structured environments such as within biofilms, within soil, or when growing in or on the tissues of plants and animals. Second, phage growth as plaques is a central technique to phage studies, yet appears to be under appreciated by phage workers in terms of its complexity. Third, selective pressures acting on phage during plaque growth differ from those seen during broth growth. In this chapter we will discuss just what a plaque is, how one forms, and what can affect plaque size. We will describe methods, both experimental and theoretical, that have been employed to study plaque growth. As caveats we will discuss why plaque formation failure is not necessarily equivalent to virion inviability (Note 1). We also will consider problems with inferring phage broth growth fitness as a function of plaque characteristics (Note 2).