Stephen T. Abedon

Phage treatment of human infections

Phages as bactericidal agents have been employed for 90 years as a means of treating bacterial infections in humans as well as other species, a process known as phage therapy. In this review we explore both the early historical and more modern use of phages to treat human infections. We discuss in particular the little-reviewed French early work, along with the Polish, US, Georgian and Russian historical experiences. We also cover other, more modern examples of phage therapy of humans as differentiated in terms of disease. In addition, we provide discussions of phage safety, other aspects of phage therapy pharmacology, and the idea of phage use as probiotics.

Phage Therapy in Clinical Practice: Treatment of Human Infections

Phage therapy is the application of bacteria-specific viruses with the goal of reducing or eliminating pathogenic or nuisance bacteria. While phage therapy has become a broadly relevant technology, including veterinary, agricultural, and food microbiology applications, it is for the treatment or prevention of human infections that phage therapy first caught the worlds imagination--see, especially, Arrowsmith by Sinclair Lewis (1925)--and which today is the primary motivator of the field. Nonetheless, though the first human phage therapy took place in the 1920s, by the 1940s the field, was in steep decline despite early promise. The causes were at least three-fold: insufficient understanding among researchers of basic phage biology; over exuberance, which led, along with ignorance, to carelessness; and the advent of antibiotics, an easier to handle as well as highly powerful category of antibacterials. The decline in phage therapy was neither uniform nor complete, especially in the former Soviet Republic of Georgia, where phage therapy traditions and practice continue to this day. In this review we strive toward three goals: 1. To provide an overview of the potential of phage therapy as a means of treating or preventing human diseases; 2. To explore the phage therapy state of the art as currently practiced by physicians in various pockets of phage therapy activity around the world, including in terms of potential commercialization; and 3. To avert a recapitulation of phage therapys early decline by outlining good practices in phage therapy practice, experimentation, and, ultimately, commercialization.

Pros and cons of phage therapy.

Many publications list advantages and disadvantages associated with phage therapy, which is the use of bacterial viruses to combat populations of nuisance or pathogenic bacteria. The goal of this commentary is to discuss many of those issues in a single location. In terms of “Pros”, for example, phages can be bactericidal, can increase in number over the course of treatment, tend to only minimally disrupt normal flora, are equally effective against antibiotic-sensitive and antibiotic-resistant bacteria, often are easily discovered, seem to be capable of disrupting bacterial biofilms, and can have low inherent toxicities. In addition to these assets, we consider aspects of phage therapy that can contribute to its safety, economics, or convenience, but in ways that are perhaps less essential to the phage potential to combat bacteria. For example, autonomous phage transfer between animals during veterinary application could provide convenience or economic advantages by decreasing the need for repeated phage application, but is not necessarily crucial to therapeutic success. We also consider possible disadvantages to phage use as antibacterial agents. These “Cons”, however, tend to be either relatively minor.

Bacteriophage latent-period evolution as a response to resource availability

ABSTRACT Bacteriophages (phages) modify microbial communities by lysing hosts, transferring genetic material, and effecting lysogenic conversion. To understand how natural communities are affected it is important to develop predictive models. Here we consider how variation between models—in eclipse period, latent period, adsorption constant, burst size, the handling of differences in host quantity and host quality, and in modeling strategy—can affect predictions. First we compare two published models of phage growth, which differ primarily in terms of how they model the kinetics of phage adsorption; one is a computer simulation and the other is an explicit calculation. At higher host quantities (∼108 cells/ml), both models closely predict experimentally determined phage population growth rates. At lower host quantities (107 cells/ml), the computer simulation continues to closely predict phage growth rates, but the explicit model does not. Next we concentrate on predictions of latent-period optima. A latent-period optimum is the latent period that maximizes the population growth of a specific phage growing in the presence of a specific quantity and quality of host cells. Both models predict similar latent-period optima at higher host densities (e.g., 17 min at 108 cells/ml). At lower host densities, however, the computer simulation predicts latent-period optima that are much shorter than those suggested by explicit calculations (e.g., 90 versus 1,250 min at 105 cells/ml). Finally, we consider the impact of host quality on phage latent-period evolution. By taking care to differentiate latent-period phenotypic plasticity from latent-period evolution, we argue that the impact of host quality on phage latent-period evolution may be relatively small.

Lysis from without

In this commentary I consider use of the term “lysis from without” (LO) along with the phenomenon’s biological relevance. LO originally described an early bacterial lysis induced by high-multiplicity virion adsorption and that occurs without phage production (here indicated as LOV). Notably, this is more than just high phage multiplicities of adsorption leading to bacterial killing. The action on bacteria of exogenously supplied phage lysin, too, has been described as a form of LO (here, LOL). LOV has been somewhat worked out mechanistically for T4 phages, has been used to elucidate various phage-associated phenomena including discovery of the phage eclipse, may be relevant to phage ecology, and, with resistance to LO (LOR), is blocked by certain phage gene products. Speculation as to the potential impact of LOV on phage therapy efficacy also is fairly common. Since LOV assays are relatively easily performed and not all phages are able to induce LOV, a phage’s potential to lyse bacteria without first infecting should be subject to at least in vitro experimental confirmation before the LOV label is applied. The term “abortive infection” may be used more generally to describe non-productive phage infections that kill bacteria.

Experimental Examination of Bacteriophage Latent-Period Evolution as a Response to Bacterial Availability

ABSTRACT For obligately lytic bacteriophage (phage) a trade-off exists between fecundity (burst size) and latent period (a component of generation time). This trade-off occurs because release of phage progeny from infected bacteria coincides with destruction of the machinery necessary to produce more phage progeny. Here we employ phage mutants to explore issues of phage latent-period evolution as a function of the density of phage-susceptible bacteria. Theory suggests that higher bacterial densities should select for shorter phage latent periods. Consistently, we have found that higher host densities (≥∼107 bacteria/ml) can enrich stocks of phage RB69 for variants that display shorter latent periods than the wild type. One such variant, dubbed sta5, displays a latent period that is ∼70 to 80% of that of the wild type—which is nearly as short as the RB69 eclipse period—and which has a corresponding burst size that is ∼30% of that of the wild type. We show that at higher host densities (≥∼107 bacteria/ml) the sta5 phage can outcompete the RB69 wild type, though only under conditions of direct (same-culture) competition. We interpret this advantage as corresponding to slightly faster sta5 population growth, resulting in multifold increases in mutant frequency during same-culture growth. The sta5 advantage is lost, however, given indirect (different-culture) competition between the wild type and mutant or given same-culture competition but at lower densities of phage-susceptible bacteria (≤∼106 bacteria/ml). From these observations we suggest that phage displaying very short latent periods may be viewed as specialists for propagation when bacteria within cultures are highly prevalent and transmission between cultures is easily accomplished.

Selection for bacteriophage latent period length by bacterial density: A theoretical examination

In bacteriophage (phage), rapid and efficient intracellular progeny production is of obvious benefit. A short latent period is not. All else being equal, a longer latent period utilizes host cell resources more completely. Using established parameters of phage growth, a simulation of three successive phage lysis cycles is presented. I have found that high, but not low, host cell densities can select for short phage latent periods. This results from phage with short latent periods more rapidly establishing multiple parallel infections at high host cell concentrations, whereas phage with long latent periods are restricted to growth within a single cell over the same period. This implies that phage with short latent periods habitually grow in environments that are rich in host cells.

Kinetics of Phage-Mediated Biocontrol of Bacteria

Bacteriophages (phages) are the viruses of bacteria. One subset of phages, those that can be described as obligately lytic, can effect an active phage therapy because their population growth occurs at the expense of bacterial survival. That is, phages can be employed to reduce bacterial loads--such as in animals preslaughter, in foods postharvest, or in humans postinfection--and in the process can actually increase what in pharmacological terms would be their antibacterial dose. This self-amplification may provide advantages if either antibacterial dosing or penetration to target bacteria is otherwise constrained. One situation in which these kinetic aspects of drug delivery may be constrained is in preslaughter treatment of food animals toward control of zoonotic pathogens (e.g., Escherichia coli O157:H7 in cattle). In such treatment, the self-amplifying nature of phages may be harnessed, though potentially under time constraints. In this discursive I cover three areas. The first is semantic, where I contrast the terms phage therapy and phage-mediated biocontrol of bacteria, both of which are employed to describe similar but perhaps not identical procedures. Second, I consider the importance of time in therapy or biocontrol procedures while contrasting passive versus active therapies. Third, I discuss conceptually how to go about modifying phage characteristics to increase rates of phage population growth and do so explicitly by casting phage infection in terms of Michaelis-Menten saturation kinetics. I conclude suggesting that phage therapy ultimately may be rationally guided by theoretical considerations of the impact of phage properties on rates of phage population growth.

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.

Bacterial ‘immunity’ against bacteriophages

Vertebrate animals possess multiple anti-pathogen defenses. Individual mechanisms usually are differentiated into those that are immunologically adaptive vs. more “primitive” anti-pathogen phenomena described as innate responses. Here I frame defenses used by bacteria against bacteriophages as analogous to these animal immune functions. Included are numerous anti-phage defenses in addition to the adaptive immunity associated with CRISPR/cas systems. As these other anti-pathogen mechanisms are non-adaptive they can be described as making up an innate bacterial immunity. This exercise was undertaken in light of the recent excitement over the discovery that CRISPR/cas systems can serve, as noted, as a form of bacterial adaptive immunity. The broader goal, however, is to gain novel insight into bacterial defenses against phages by fitting these mechanisms into considerations of how multicellular organisms also defend themselves against pathogens. This commentary can be viewed in addition as a bid toward integrating these numerous bacterial anti-phage defenses into a more unified immunology.