Abstract:  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.

(2010) additionally distinguish between phage banks for the sake of research and the maintenance and employment of phage banks at the point of phage application (p (2010) and Abedon et al (2010) consider in terms of bacterial mechanisms of resistance to phages (2010), p (2011) (2011) , 1983; Weber-Dabrowska et al , 1984) , 2000) , 2001), and a number of authors have argued that use of phage banks may be important to the general success of phage therapy as a broadly adopted antibacterial strategy (Ackermann and DuBow, 1987; Gill and Young, 2011; Kutter, 2005; Petty et al , 2005) , 2005; Kutter et al , 2005; Rountree, 1949) , 2006) and diabetes treatments (Triplitt, 2006) , 2007) , 2007) , 2007) or at least as a backup to cocktail use (Kutter, 2009); see also (Nishikawa et al , 2007; Rhoads et al , 2008) , 2008; Skurnik et al , 2008; Slopek et al , 2009) , 2009) , 2009) and there is little evidence that this is not also so when phages are taken in combination with other phages , 2009; Guttman et al , 2009; Wright et al , 2010) , 2010) , 2010) , 2010) , 2010) , 2010) include necessary delays in determining bacterial phage susceptibility before treatment, a requirement for phage susceptibility testing expertise, and generally a need to characterize as well as store and/or amplify more Phage Therapy Pharmacology: Phage Cocktails 15 Authors personal copy phage isolates than is necessarily the case when instead employing preformulated phage cocktails , 2010): This amounts to producing complex therapeutic cocktails consisting of phages perfectly matched in terms of life cycle properties (p , 2010; McVay et al , 2010; Wright et al , 2011; Curtright and Abedon, 2011) , 2011; Curtright and Abedon, 2011) , metabolism) and that different phage types could vary in terms of other pharmacokinetic aspects , phage susceptibility type) [In contrast] cocktails may be used because bacterial resistance is less likely to develop, presumably resulting in a product more likely to be otherwise efficacious Considering the same logic, but from the opposite perspective, cocktails can be seen as seeking equivalent benefits as a phage bank approach, in terms of the potential diversity of phages available for application, but with parallel empiricism replacing serial experimentation In this chapter, however, we tend to avoid using this term, employing cocktail instead, with cocktail meaning, unless we indicate otherwise, more than one phage type (¼polyphage) rather than one or more phage types used in combination with some other, nonphage-active ingredient We discuss phage banks further after considering, in the following section, the terms monovalence and polyvalence along with their relationships to the concept of phage host range 0 of 0 0001 five times would still retain 0 12 Benjamin K 163) 18 Benjamin K 5: Whether continuously replenished or otherwise static, a phage bank is a collection of phages that have been isolated, characterized to some extent, and are available either as phage preparations or, instead, as yet-unexpanded phage stocks, for matching to a particular recently isolated target bacterium 5: Cocktails may be advantageous for regulatory reasons in that a set of phages may be marshaled through the regulatory process (although it should be noted that this has not been a concern in the OmniLytics case), or in order to avoid a direct interface between user and producers (and thereby avoid the shipping of infected samples back to phage suppliers) 50, considering phage banks especially from the perspective of the biocontrol of bacterial pathogens of plants: General criticisms against the phage bank approach is that it is not consistent with either presumptive or prophylactic phage use (that is, phage application prior to bacterial identification), it requires additional expertise from users (who would then not only apply phages but also identify phage targets), and requires facilities for either long-term phage storage or rapid amplification of specific phage strains to high quantities 53) 55 (Balogh et al 8 Benjamin K 9995 of the original (with quotes to indicate that these numbers are for illustrative purposes only) a A A A A A A battery of phages displaying dissimilar host ranges, however, also could display substantially different performances in terms of adsorption rates, burst sizes, and latent periods A further consideration is that just as cocktails of phages may be thwarted in their ability to target low densities of phage-resistant bacteria, particularly given active treatment, these concerns should be even greater if one is relying on in situ phage evolution to supply resistance-countering phages (Kysela and Turner, 2007) A monophage thus is unambiguously a single phage type A potentially related issue is that the individual components of phage cocktails, if each displays a narrow host range, will select for phage resistance against that specific phage in only a narrow subset of exposed bacteria A second example appears to be phage P100, which has been shown to be bactericidal against 95% of 250 different foodborne Listeria spp A Utah bacteriophage company, OmniLytics, however, currently provides phages to farmers, commercially, that have been custom matched to plant pathogenic bacteria (Gill and Hyman, 2010) A variety of phage and bacterial characteristics serve to limit phage host range to less than the totality of bacterial types available, and in fact phage host range can be limited to collections of related species, individual bacterial species, or even, as appears to be often the case, only a subset of individual strains of bacteria making up a single bacterial species (Hyman and Abedon, 2010) Abedon Authors personal copy adversely impact potentially beneficial bacteria Abedon Authors personal copy application of multiple phages in parallel is just another means of saying as phage cocktails Abedon Authors personal copy bacterial phytopathogens Abedon Authors personal copy C Abedon Authors personal copy Combination therapy can still be useful if there is utility to avoiding delays in the application of effective treatments Abedon Authors personal copy employed only locally Abedon Authors personal copy in the laboratory, prior to phage use therapeutically, can be less than maximally rigorous Abedon Authors personal copy terms bacteriophage, describing the viruses particularly of domain Bacteria, along with macrophage, are the most familiar Abedon Authors personal copy V Absent such targeting information, therapy can be described as presumptive or empirical Absorption describes the movement of drugs into the blood as can occur via various routes including orally (per os) Absorption is not a dominant issue for phage treatments that involve, instead, only local treatment Active penetration into bacterial biofilms Mixed coinfections could be problematic given a requirement for active phage replication during the treatment of spatially constrained bacteria, such as bacteria inhabiting biofilms Active therapy even with phage cocktails thus may be inherently incompatible with early interference with the evolution of bacterial resistance to phages Additional problems with combination therapies can be increased costs and decreased convenience, the latter to the extent that multiple drugs cannot be administered simultaneously Additionally, one can apply phages repeatedly, that is, in multiple or even continuous doses, so that even if infection productivity is constrained, additionally supplied phages still might penetrate into spatial bacterial structures Again, this is less of an issue for phages that are 4 Benjamin K All these concerns are seen with multidrug anti-HIV treatments (Carr, 2003; Schackman et al All these considerations—diversity of targets as well as conditions, requirements for substantial eradiation, time pressure, and formulation development issues—can lead to the use of phage cocktails rather than individual phages in the course of phage therapy Alternatively, lysis from without or high-multiplicity-associated abortive infections (Abedon, 2011b) could produce similar concerns even with monophages Alternatively, phage bank approaches to phage distribution (this section) can be quite successful even when not supplying cocktails or polyvalent phages Alternatively, rare bacterial types, even if left untreated, may not supply much of a health threat (Levin and Bull, 2004) Alternatively, such incompatibilities may be assumed to generally be present within cocktails and addressed in the course of development of application strategies Another consideration is that a phages host range for passive treatment purposes would describe, at a minimum, its bactericidal activity Another consideration is that while a single phage may be able to kill a diversity of bacterial targets in the laboratory, that same phages ability to reproduce while infecting the same diversity of bacterial hosts could be more constrained, as may be distinguished in terms of bactericidal versus productive host ranges, as discussed above Another way of saying this is that if Phage Therapy Pharmacology: Phage Cocktails 9 Authors personal copy individual reductions in health are expected to be small, then overall reductions, in combination, too should not be large, for example, a reduction from 1 Another way to put this is that it can be tempting, especially for reasons of easing regulatory approval, to generate phage cocktails based upon a single, well-characterized, platform phage such as phage T4 (Pouillot et al Antagonistic drug–drug interactions can also occur, that is, in terms of drug positive pharmacodynamic effects (efficacy) and/or pharmacokinetic effects rather than debilitating side effects Antibacterials that are approved for application to human tissues also must display a selective toxicity As a consequence, substantially greater clinical expertise may be required of the provider given the use of phage banks As a consequence, the phage potential to replicate within the presence of target bacteria can be considered to be a property, pharmacokinetically, that is equivalent to drug metabolism As applied to phage therapy, one must define drug more broadly than simply small-molecule agents (Abedon, 2012) As applied to treat cancers or infectious diseases, combinations typically are of drugs that possess different mechanisms of action so that mutations that result in resistance do not individually block the function of more than one drug, that is, such that cross-resistance is unlikely At least as typically employed, phage banks instead involve phage use following bacterial testing for phage susceptibility At least one drug of a combination, that is, should retain activity against newly drug-arising pathogens, blocking further growth and therefore resistance evolution B B B B Bacteria targeted by phages in phage therapy can consist of single clones, can contain mutations to phage resistance, can vary physiologically, can be difficult for phages to physically reach, or can consist of mixed infections, with different phage isolates potentially displaying different therapeutic abilities as a function of these variables Broadening phage formulation spectra of activity can be advantageous, that is, for reasons that can be more apparent under conditions where it is not possible to personalize phage choice against specific bacterial infections By contrast, if monophages making up cocktails differ substantially in their infection characteristics, then the likelihood of complete failure due to poor matches between phages, bacteria, bacterial physiologies, and/or infection conditions might be lower C C C Chan and Stephen T Chan and Stephen T Chan and Stephen T Chan and Stephen T Chan and Stephen T Chan and Stephen T Chan and Stephen T Chan and Stephen T Chan and Stephen T COCKTAIL-RELATED TERMS Early use of the word cocktail was in reference to mixed alcoholic drinks Cocktails, if reasonably well formulated, can have the greatest potential of all Combination phage therapy, similar problems Mixing of phages into cocktails is not expected to additively decrease the overall safety of phages to a substantial extent since individual phage isolates, on their own, do not display substantial negative pharmacodynamic effects (Abedon and Thomas-Abedon, 2010; Abedon et al Combination therapy is simply the simultaneous use of multiple drugs or therapies for the treatment of a single condition COMBINATION THERAPY The formulation of phages into cocktails is an example of drug combination therapy, also known as polytherapy Combination therapy, benefits Most prominently, the benefits of combination therapy are that the use of two or more independently acting agents should substantially decrease the likelihood of evolution to resistance Combination therapy, costs Problems with combination therapy also exist CONCLUSION The narrow host range of phages during use as antibacterial agents presents a double-edged sword Consistently, phage cocktails can be described as consisting of a combination of more than one monophage D D Depending on the phage as well as the bacteria targeted, this process can require solely phage-mediated bacterial killing (so-called passive or inundative treatment) or, instead, demand phage population growth, in situ, to generate sufficient phage densities to achieve inundation (so-called active treatment); see Abedon and Thomas-Abedon (2010) for review of these concepts Drug development issues The utility of using cocktails goes beyond a potential to initiate treatment prior to precise diagnosis of etiologies e Each of these properties will tend to vary as a function of phage genotype, target bacterium genotype as well as current physiology, and environmental circumstances Equivalently, there is a need in phage therapy development for a weeding out of ineffective phages (which may do well in vitro but have little or no effect in vivo) p Even if phages have been somewhat thoroughly characterized, phage performance in vivo can fail to match phage performance in vitro (Rivas et al Excretion is movement of drugs out of the body in the same form as they were administered Extended host-range breadth, that is, could be a consequence of either (1) phages recognizing multiple receptors that, for example, are different between different bacterial species, or strains, that is, different receptors on different bacteria (Morona and Henning, 1984), or (2) phages recognizing equivalent phage receptors that are found on different bacterial species, that is, same receptors, different bacteria (de Vries et al Following absorption, distribution is a drug movement into nonblood tissues For a nice overview of the formulation of phage cocktails and their use, along with expansion of phage host ranges in general, see Goodridge (2010) For active treatment, however, a phages virion-productive host range is also important For discussion of the advantages of phage therapy for the treatment of bacterial infections, particularly relative to the use of traditional antibiotics, see Loc-Carrillo and Abedon (2011) For example, in the treatment of mental illnesses such as bipolar disorder, drug combinations can result in increases in side effects (Ketter, 2009) such that it can be unclear whether combination therapy provides net benefits (Geddes et al For example, mixing bacteriostatic and bactericidal drugs can result in reduced effectiveness, particularly of the bactericidal drug (Johansen et al For example, such host-range breadth appears to be the case with Staphylococcus phage K (OFlaherty et al For example, the combination of streptomycin and penicillin, as often used in cell culture to reduce contamination by bacteria, is efficacious particularly in light of their synergistic bacterial killing action (Plotz and Davis, 1962) For example, the koala (Phascolarctos cinereus) has a monophagous diet that consists of eucalypt leaves, and the giant panda (Ailuropoda melanoleuca) has a diet that consists almost exclusively of bamboo For more general reviews of phage therapy, such as of humans, see Kutter et al For phages, however, the definition of monophage is subtly different from the usage considered in the previous paragraph and this reflects use of the term phage as a noun (as in bacteriophage) rather than as a verb or adjective For references to earlier phage therapy reviews, see Abedon and Thomas-Abedon (2010), and for consideration of phage use as antibiofilm agents, see Abedon (2010, 2011a) Formulation of phage cocktails thus can achieve a broadening of spectra of activity but there is no set, simple, or necessarily ideal way by which spectrum of activity, even as an equivalent to phage host range, may be defined (Hyman and Abedon, 2010) From Balogh et al From Gill and Hyman (2010), p From Gill and Hyman (2010), p Further, bacterial clearing can occur as an only indirect consequence of phage presence, such as if endolysins or bacteriocins should contaminate phage stocks, potentially negating even evidence of phage bactericidal activity Further, it is important to keep in mind that phage banks, though they can be used as an alternative means by which phages are identified for use versus the employment of predefined phage cocktails, in fact can also be employed to develop phage cocktails within a context of personalized medicine Further, the phage spectrum of activity, that is, what bacteria they affect, tends to be more limited than is the case with typical, clinically available chemical antibiotics Further, though lowering phage densities per dose could be a rational response to such observations, supplying phages in multiple doses over the course of treatments also should be considered Given close coordination between clinics and microbiology labs, it should also be possible to switch to different phage isolates given an observation in infections of resistance evolution to phages Here, productive phage infection may be required to deliver phages deeper into biofilms Here, we consider various issues relevant especially to such formulation of phages as cocktails for phage therapy, including in comparison to alternative strategies of dispensement Here, we have provided a discussion of the utility and limitations of phage use as cocktails Host range is a description of the types of organisms another organism can successfully infect or colonize (Chan and Abedon, 2012) I Ideally, those phages making up phage banks will have been tested, prior to use, in terms of safety, such as by ruling out the potential to display lysogenic infections or otherwise encode bacterial virulence factors, that is, by limiting phage use particularly to professionally lytic isolates (Curtright and Abedon, 2011) If a cocktail is employed rather than a broader host-range phage, then the breadth of circumstances under which phage formulations may be effective against the same bacterial strain might be broadened If different phage types within a cocktail possess different potentials to replicate in situ, however, then phage coinfections may no longer be mixed by the time intermediate or higher phage multiplicities appear II III Importantly, the clinician retains the potential to employ different phages should the initially chosen isolates prove to be less efficacious than desired In addition to inactivation, metabolism can activate drugs, particularly prodrugs In addition, a number of reviews of phage therapy pharmacology have been recently published (Abedon, 2009a,b, 2011c, 2012; Abedon and Thomas-Abedon, 2010; Curtright and Abedon, 2011) In addition, if those phage-resistant bacteria do replicate to sufficient densities that they can support active treatment, then cocktail use against them will be equivalent to the primary treatment of bacteria displaying alternative phage susceptibilities In addition, in certain instances drugs can act synergistically which, for antimicrobial or anticancer drugs, can be envisaged as one drug weakening the target in some manner so that the impact of the second drug, on the target, is enhanced In addition, the economics as well as regulation of phage formulations, and their development, can increase the desirability of not limiting what bacteria are targeted during treatments In addition, we are unaware of evidence that phage use might impact the effects associated with other drugs that an individual might be taking or of safety issues that could stem from immune responses to phage cocktails versus individual phage isolates In certain cases, antibiotic combinations also can more rapidly diminish the presence of target bacteria, reducing the time during which resistance can arise (Torella et al In circumstances where the cost of the phage material is large relative to other costs, then having more phages per formulation could be relevant In clinical medicine, however, other costs can be so high, including the cost of treatment failure, that it is unlikely that increases associated with combination therapy will have a marked impact on the economic utility of phage use In doing so, the full advantages of employing cocktails versus monophages as phage formulations, particularly in terms of diversity of phage-infection physiological properties, may not be as fully realized, as indeed Pouillot et al In either case, however, note that phage isolation, and characterization, are effected well prior to the point of therapeutic application In general, cocktails may be preferable for the sake of efficacy (hitting bacteria with more than one phage type), whereas the phage bank approach can be preferable for the sake of phage host specificity (direct matching of phages with specific target pathogens) In light of these various contributors to target diversity, a single phage type may not be suitable or, alternatively, may not be sufficient to achieve desired levels of bacterial eradication In other words, just because enough phage types are present within a cocktail to cover possible phage-resistant bacterial phenotypes, there is no guarantee that the necessary phages will be present at sufficient densities if active treatment is being relied upon In particular, phage often suffers from an ambiguity in plurality, with phage and phages often and perhaps unfortunately employed identically even if reference is being made to multiple phage types (Ackermann, 2011) In particular, if phages are added at relatively low densities, that is, so as to effect an active treatment, then the ability of phages with the necessary host range to encounter minority populations of phage-resistant bacterial mutants may be somewhat limited In particular, phages in vitro are often cultured on bacteria under somewhat optimal conditions for growth, which could differ dramatically from in situ circumstances In particular, when a phage is intended to target a single bacterial species, then neither polyvalent monophages nor use of phage cocktails may be necessary, at least so long as that phages host-range breadth within that bacterial species is quite wide In terms of clinical trials, the company Ampliphi (formerly Biocontrol) is a leader in this approach to phage formulation development, which in their case at least initially has been against Pseudomonas infections (Hawkins et al In this section, we consider pharmacological issues relevant to employing phage cocktails In this section, we consider the costs and benefits of phage formulation into cocktails from the perspective of the use of combination therapies in general In this section, we ponder further the relationships between cocktails, phage banks, and phage polyvalence, beginning with discussion of phage banks specifically In this section, we provide an exploration of additional terms, particularly the etymology of phage along with the use of the terms monophage, polyphage, and phage bank Including more phage types, though, can also increase the need for phage replication in situ if mixing phages into cocktails reduces densities of individual phage isolates within formulations Indeed, an argument for antibiotic substitution with phages for economic reasons in at least certain cases can be made (Miedzybrodzki et al Indeed, antibiotics, though chosen for human use in part based on their selective toxicity, have–perhaps especially for economic reasons–tended to possess relatively broad spectra of activity that can include substantial numbers of nontarget bacteria (Fischbach and Walsh, 2009; Hawrelak and Myers, 2004) Indeed, phage cocktails, as a form of antibacterial combination therapy, likely have additional antiresistance evolution aspects, as we now consider Indeed, phage treatment of bacterial infections that likely contain bacterial biofilms can routinely involve the use of phage cocktails containing relatively low phage densities, that is, _108ml, in combination with multiple or continuous dosing (Abedon, 2012; Kutter et al Information that can be lacking includes knowledge of the genus, species, strain, or, perhaps especially, the degree of bacterial susceptibility to specific phages (a Instead, over the course of in situ phage population growth, cocktails may to some degree be reduced, due to natural selection, to monophages (Cairns and Payne, 2008) INTRODUCTION A bacteriophage, or phage for short, is a virus whose host range consists of members of domain Bacteria, which includes all known pathogenic prokaryotic organisms (Gill and Brinkman, 2011) isolates It also allows the development and marketing of phage formulations for treatment of specific bacterial species, particularly with a higher potential for antibacterial efficacy given the inclusion of greater numbers of phages It is entirely possible, therefore, if using near monocultures of phages that differ solely in adsorption properties, to inadvertently employ a phage type that performs well in the laboratory but poorly under specific in situ circumstances or against specific bacterial targets It is possible that mixed coinfections could interfere to some degree in the transition between these two states, low versus high multiplicities IV k Laboratory characterization of phage performance Phage performance in the laboratory may not always match phage performance in situ Last, target bacteria can differ, perhaps substantially, from the bacterial hosts against which phages may have been characterized in the laboratory Limitations on ability of diverse phages to find phageresistant bacterial mutants An additional consequence of spatial structure can be limitations on phage cocktail ability to hinder bacterial evolution to phage resistance LIMITATIONS ON COCKTAIL UTILITY While it is facile to envisage phage cocktails as being inherently more effective against specific bacterial populations in comparison to monophages, in fact there exist two basic limitations on this potential that could impact the design of phage therapy treatments Metabolism typically describes drug chemical modification, though it more loosely can include drug modifications in general such as immune-system-mediated inactivation Mixed coinfection impact on infection productivity Different phage types may not be compatible during coinfection, resulting in reduced infection robustness by one or more of the infecting phages (Abedon, 1994) Mixed coinfections also may not result in substantial problems given active treatment of bacteria suspendedwithin wellmixed fluids Monophage The term monophagy, associated with ecology, refers to an extreme in narrowness or specialization of diet which, in this case, consists of only a single resource such as a single species of plant or animal MONOVALENCE, POLYVALENCE, AND PHAGE HOST RANGE Monovalence and polyvalence—contrasting monophage and polyphage— are qualitative descriptions of phage host ranges More generally, pharmacokinetics can be described as the bodys impact on drugs More precisely, a monovalent phage can recognize only a single receptor type, as found on the surface of bacteria, and a polyvalent phage may be able to recognize more than one receptor type, with recognition in both cases in terms of the phage–bacterial interaction that occurs at the point of initial phage contact with a bacterium Moreover, whether phages are employed as monophages or instead are mixed into cocktails, the real distinction among clinical strategies is between the use of phages in a more personalized context versus fixed formulations designed for much broader application Negative pharmacodynamics are side effects associated with drug use, which to a limited extent we also consider Note that Balogh et al Note that in the case of phage P100, again as an example, the majority of effectiveness, in this case upon the treatment of foods, is expected to be achieved through inundation, that is, passive treatment (Hagens and Loessner, 2010) Note that phage banks are also maintained in the fermented food industry as a record of potentially fermentation-disruptive phages against which resistant bacterial strains can be selected (Sanders, 1987) Note that some overlap between these categories can exist since it is possible, as noted, to generate phage cocktails on a per-patient basis from phage banks (Leszczynski et al On the one hand, body tissues along with potentially beneficial bacterial generally are unaffected by phage therapy On the other hand, a phage bank employs a more customizable approach to phage therapy On the other hand, drugs that are inherently safer can be more versatile including in their use in combination with other drugs Once all bacteria are infected, that is, once high multiplicities of phage adsorption have been attained following phage population growth, then mixed coinfections no longer will matter so long as those infections remain bactericidal One consideration is that if a single phage displays too wide a host range, then its impact on nontarget bacteria may be greater, a situation which could potentially be mitigated by employing multiple, narrowly targeted phages, that is, as cocktails One means of addressing these complications could be to test for phage incompatibilities in laboratory culture One such strategy is to rely on, when treating biofilms with phages (Abedon, 2010, 2011a), not just active penetration but active therapy in general, that is, with the above-conjectured in situ natural selection reducing cocktail diversity prior to the occurrence of mixed coinfections by phages One then determines, for each infection to be treated—including against multiple species if need be—which phage or phages are best suited to treat the apparent etiologies Operationally, either of these cases could be described as examples of polyvalency though strictly only the first represents the actual recognition by a phage of substantially different receptors Our general advice, therefore, is to perform dose–response curves when developing phage therapy protocols and to not be too surprised should relatively high doses interfere especially with the productivity of mixed coinfections nor consequent phage penetration into bacterial biofilms Our overall conclusion is that employing phage cocktails rather than monophages may be useful for reasons of economics, for gaining regulatory approval, for convenience, or in terms of speed of delivery, though we caution against overselling their potential beyond simply broadening the spectrum of activity of formulations Our usage here is in the latter sense Phage bank With presumptive treatment, to at least a first approximation, the wider an antibacterial drugs spectrum of activity the greater the likelihood of a pathogens inclusion in that spectrum PHAGE BANKS Identification of phages displaying substantial host-range breadth might simplify phage therapy development Phage banks thus represent one means toward a pathogen-directed approach to phage therapy, one which, in turn, can be viewed effectively as a form of personalized antibacterial medicine (Kutter, 2009) Phage banks, therefore, are unlikely to represent the first phage therapy strategy to be broadly exploited within the context of modern Western medicine Phage burst sizes or latent periods might be smaller or longer in vivo, thereby slowing the rates or magnitude of phage population growth, which can be a concern especially if active treatment is anticipated (Abedon, 2012) Phage cocktails, which consist of more than one phage type, thus can be described instead as a polyphage or polyphage formulations Phage host range, at a minimum, can be varied solely in terms of phage adsorption characteristics (Goodridge, 2010) Phage Literally meaning to eat or devour, in Greek (jageI˜ n), the term phage, including as a suffix, has been applied to numerous phenomena in which these actions occur; see too the suffix, phagy Phage P100, in addition, appears to be polyvalent at least in the sense that it can be propagated on Listeria innocua (Carlton et al Phage spectra of activity A phages potential to impact a bacterial population is dependent to a large extent on its host range (Chan and Abedon, 2012; Hyman and Abedon, 2010) Phage therapy along with the related phage-mediated bacterial biocontrol is the use of phages to reduce or eliminate nuisance or pathogenic bacteria from bodies or environments (Abedon, 2009b); see also, for example, Abedon et al PHAGE THERAPY AND PHARMACOLOGY Phage therapy is the application of phages to infect and kill unwanted bacteria Phage therapy pharmacology Pharmacology is the study of drug–body interactions with the goal of developing safe, effective, and otherwise useful pharmaceuticals Phage Therapy Pharmacology: Phage Cocktails 17 Authors personal copy B Phage Therapy Pharmacology: Phage Cocktails 19 Authors personal copy On the other hand, individual phages, that is, monophages, tend to be limited in their potential to be used presumptively against bacterial infections Phage-resistant mutants similarly, if newly present within a bacterial population, will not be expected to be present at sufficiently high densities to support phage amplification to densities sufficient to control those populations Phages may not adsorb with the same efficiency in vivo due to differences in the chemical composition of the adsorption environment (Hyman and Abedon, 2009) Phages, despite their status as viruses, not only display selective toxicities, impacting target bacteria but not our own tissues, but also, in practice, have been found to be quite safe (Abedon and Phage Therapy Pharmacology: Phage Cocktails 5 Authors personal copy Thomas-Abedon, 2010; Abedon et al Phages, that is, can be remarkably safe pharmaceuticals (Bruttin and Bru¨ ssow, 2005; Rhoads et al Pharmacodynamics describes a drugs impact on bodies Pharmacokinetics are those things that impact the ability of a drug to reach concentrations, within the vicinity of target tissues, that are required for the achievement of positive pharmacodynamic effects Pharmacology is also typically differentiated into what is described as pharmacodynamics versus pharmacokinetics Polyphage Contrasting monophagy, ecologically the term polyphagy refers to the consumption of more than one food item, that is, having a varied diet and/or being less specialized in terms of what resources a species exploits Phage Therapy Pharmacology: Phage Cocktails 11 Authors personal copy for food Positive pharmacodynamics for antibacterial agents thus describes the successful treatment of bacterial infections Problems with phage banks The downsides to the phage bank approach (Balogh et al productive host range; previous section) Recall that the ability of phages to replicate can be described as a pharmacokinetic property (i Resistance evolution concerns, which potentially could reduce the useful lifespan of an antimicrobial cocktail, therefore could be inherently lower, even with phage cocktails, in comparison to broaderspectrum antibacterials Roughly, a phage that is described as monovalent has a narrower host range than one that is polyvalent So-called spot testing in particular, where phages are applied in large numbers to an immature bacterial lawn to visualize bacteria-killing ability (Carlson, 2005), is not always a good indicator especially of phage replication ability (Abedon, 2011b) Specific etiologies can also be less than fully identified prior to the initiation of antibacterial treatment Such a strategy can be viewed as a means of obtaining the utility of cocktails in serial rather than in parallel, that is, where the 14 Benjamin K Such concerns may be overcome by supplying phage doses that are directly inundative (Cairns and Payne, 2008) Such longevity as well as broad applicability will be crucial to the extent that every phage found within a phage cocktail, or even just every phage cocktail, must be subject to full and independent regulatory approval Such secondary treatment may be possible particularly if phages are supplied either in multiple doses or continuously Such testing, however, can be time-consuming, may give results that vary with different hosts, and otherwise could serve as an additional limitation on what phage types should be included in a given cocktail, including in terms of how many phages ought to be included (the more phage types present, the greater the likelihood of incompatibility between phages) That is, they must not be so disruptive of body metabolism, at effective densities, that they substantially harm us The cost of development as well as production of phage pharmaceuticals could increase given greater numbers of phage types per formulation The difference between phage banks and formally developed phage cocktails, in fact, can be viewed in terms of the contrast between clinically modifiable versus off the shelf (respectively) The equivalent concept with regard to antibacterial drugs is spectrum of activity The idea with a phage bank is to obtain some number of phages that, ideally, display differences in at least host range The latter includes host chemical, immunological, and anatomical factors that can impact phage movement, phage adsorption to bacteria, and phage infection once adsorption has occurred The latter may be especially problematic due to the especially regional and seasonal nature of 16 Benjamin K The latter occurs because cocktails represent the phage equivalent of a multidrug approach to treatment The latter, of course, is a type of phagocytic cells which, in turn, are named on the basis of their ability to phagocytise (consume, eat, devour) small pieces of material, including bacteria The more phage types that one employs against a given bacterial strain during phage therapy, the more likely, in simple probabilistic terms, that at least one phage will be effective at reaching target bacteria, replicating in the course of infecting those bacteria, or just succeeding in killing them The phage bank approach, by contrast, is ill-served by this perspective of presumptive treatment The phrase bacte´riophage obligatoire is the actual first usage and indeed is the only way dHe´rrelle used the term in the original publication (dHe´relle, 1917, 2011) The primary advantage comes from a tendency for therapeutic phages to not 2 Benjamin K The primary challenge, on the other hand, is that the utility of specific phages usually is limited to only a small number of bacterial targets The reason for this is that the necessary host-range mutant phage types will be present in even lower densities than the phages explicitly found in cocktails The reasons for this are multifold The relatively narrow host range of bacteriophages presents both advantages and challenges to phage therapy The same is more or less the case for bacteriophages, where host ranges do not often span more than a single bacterial genera and, typically, a given phage is only able to infect a fraction of the strains making up a single bacterial species (Hyman and Abedon, 2010) The specific scenario can be described as an active penetration of phages into bacterial biofilms (Abedon and Thomas-Abedon, 2010) The term penetration has been used instead to describe local phage movement into body tissues as well as into bacterial biofilms (Abedon, 2012) The terms monovalent and polyvalent too are addressed, though in a separate section The trivial reason is that phage testing 6 Benjamin K The use of polyvalent or at least wide host-range phages, instead of cocktails, may not be without cost The word phage, used alone, is a shortened, essentially slang version of bacteriophage, one that has long been in common usage including in technical writing There are at least two reasons for why this should be, one trivial and the other more profound There is also no reason to expect that the combined impact of phages might result in dangerous synergies Therefore, its bactericidal host range presumably is more pertinent than its productive host range These abilities, as noted, can vary as a function of not only just bacterial genotype but also of environmental, that is, body conditions These are (1) presumptive treatment using a narrow host-range monophage, (2) presumptive treatment using a broader host range or indeed polyvalent monophage, (3) treatment using predefined cocktails (using either presumptive or pathogen-directed approaches), or (4) using a phage bank These are (1) the potential for mixed phage coinfections of individual bacteria to negatively impact phage-infection productivity (such as in terms of burst size) and (2) limitations on the ability of cocktails to inhibit bacterial evolution to phage resistance These considerations are speculative, as the detailed kinetics of phage action particularly against bacterial biofilms has not been well worked out These differences, further, have the potential to modify the ability of different phages to successfully eliminate specific target bacterial populations These impacts typically are differentiated into what are known as absorption, distribution, metabolism, and excretion These include increased side effects that stem from drug–drug interactions or increased risk of side effects in general because a patient is being exposed to more than one type of drug These latter consequences of drug use include toxicities of various kinds to body tissues as well as, in the case of antibacterial drugs, depletions of potentially beneficial bacteria resulting in bacterial dysbiosis (Hawrelak and Myers, 2004) These same concerns may also be seen even in the absence of spatial structure so long as those phages within a cocktail that are amplified in situ, that is, in the course of active treatment, are not the same phages to which bacterial phageresistant mutants are sensitive (Cairns and Payne, 2008) These targeting concerns are larger to the extent that complete bacterial eradication may be necessary or, instead, given time constraints, may Phage Therapy Pharmacology: Phage Cocktails 3 Authors personal copy limit the duration over which treatment can be attempted, such as against acute, life-threatening bacterial infections This approach also has the potential of allowing longer term utility for a given formulation as well as broader geographical use if different strains of target bacteria exist in different regions This approach is often used in the treatment of diseases that possess a high probability of resistance evolution to any one drug, as is the case with HIV as well as cancers, or in situations where the susceptibility of target tissues is not well defined, as is the case in the treatment of mental illnesses such as bipolar disorder and schizophrenia This basic approach is how phage therapy is typically implemented such as by the Wroc?aw, Poland, phage therapists (Fortuna et al This concept of polyvalence is muddled by the traditional means by which polyvalency has been established: observation of phage productivity on more than one bacterial species This concern may be less relevant if treatment is purely passive, since under such circumstances phage infections need only be bactericidal This determination at least initially takes place using in vitro assays but can continue into the treatment phase with replacement from the phage bank of phages that appear to be ineffective, that is, with new phages that possess different antibacterial properties This effect is limited, however, to situations where more than one drug in fact possess activity at the commencement of treatment This includes against diverse infection types (same bacterium but different disease states) or where active treatment is desired (bactericidal vs This is because the probability of two independent resistance mutations occurring within the same organism generally is lower than that of only a single mutation This is for similar reasons to the relative lack of concern given purely passive treatment, though the explanation is more complicated This is illustrative of what can be an important theme with combination therapy development, that is, tradeoffs between positive and negative pharmacodynamic effects, where negative effects can lead to substantial debilitation of patients, particularly in the elderly (Borchelt, 1995) This is usually accomplished via the kidney and has only a minor role in most phage therapy protocols This issue should not be a concern with phage P100, to continue the above Phage Therapy Pharmacology: Phage Cocktails 13 Authors personal copy example, so long as loss of Listeria-related normal flora bacteria of treated foods, that is, that might be affected by this phage, is not of concern This latter issue is our primary consideration in this chapter This practice dates to at least the early nineteenth century (A Subscriber, 1806) and, of course, is still in use today This usage, it should be pointed out, is not quite identical to that of the term phage This use of only a single phage type could simplify phage therapy development even if presumptive treatment is anticipated, but only, of course, if a suitable monophage may be found Though not necessarily requisite, it is implied that the components mixed as a cocktail are either more or differently effective than the components by themselves Three key variables in particular are a therapeutic phages ability to reach target bacteria, their ability to kill those bacteria once they have reached their vicinity, and, for the sake of successful active treatment, the ability of phages to replicate their numbers in situ to inundative densities Thus, a monophage refers to a single phage isolate, type, or strain (Sulakvelidze and Pasternack, 2010); for additional examples, see also (Abedon, 2011a; Go´ rski et al Thus, even if a handful of individual target bacteria, viruses, or cancer cells mutate to resistance to one applied drug it is unlikely—absent cross-resistance—for subsequent mutations to accumulate that result in resistance to all of the drugs employed Thus, high titer treatment of bacterial biofilms with phage cocktails possibly could be less effective than treatment at lower phage densities or, instead, using monophages Thus, narrow host-range monophages would have the least potential of successfully treating a given pathogen presumptively whereas wider host-range monophages would or at least could have a higher likelihood Thus, phage banks may be a less desirable approach, especially given the speed with which bacteria multiply and can infect the plant host Thus, the use of the word cocktail in reference to a mixture of therapeutic bacteriophages seems appropriate Thus, were a phage cocktail consisting of, for example, 10 phages Phage Therapy Pharmacology: Phage Cocktails 7 Authors personal copy to have an overall identical spectrum of activity to an equivalent chemical antibiotic, the number of bacterial species exposed to individual phage types, and therefore which could have the potential to evolve antiphage resistance mechanisms, would be only as wide as the host range of each individual phage Thus, when treating a body, even if the bacterial target is the same, it is possible for some phage types to be more effective under certain conditions, for example, such as penetration into infected wounds or in terms of resistance to elimination from the body, whereas other types may be less effective Thus, while a single phage possessing a wide host range—as determined, for example, by spot testing (Carlson, 2005)—may be adequate for passive, that is, inundative therapy, it may be less broadly effective if active treatment instead is sought To address this issue, monophages may be formulated into mixtures known as cocktails To augment this spectrum of activity, it is possible to mix phages that possess different host ranges To biomedical workers, the 10 Benjamin K Two quotes serve to emphasize the caveats associated with the phage bank approach to phage therapy Use of such phages rather than cocktails, however, might not be without compromise such as in terms of other aspects of phage utility Using phage banks There exist four basic approaches to phage-based antibacterial treatments VI VII VIII We provide this information both for background and to supply context for subsequent discussions What all of this has to do with phage cocktails is that polyvalency, or simply wide phage host ranges, can, to some degree, reduce the need to employ phage formulations that are cocktails (Gill and Hyman, 2010) When more than one phage type is employed per formulation, then the likelihood that all phages employed will be ineffectual, despite effective performance in the laboratory, presumably will be reduced While dietary specialization/ monophagy among large animals is relatively rare, for parasitic organisms monophagy is somewhat typical, with parasites and pathogens usually specializing on one or at most only a few host types While formulation of phages into cocktails follows a tradition in antimicrobial development of creating products with broad applicability, current trends toward more personalized medicine could favor, for reasons of improved efficacy, instead a custom tailoring of cocktails to specific pathogens present in individual patients With active treatment, the ratios of phage densities to those of target bacteria are low at the point of phage application, that is, <1, so coinfection should be relatively rare With antibacterial drugs, generally the term body must be defined more broadly as well to include associated microbiota With greater numbers of phage isolates along with greater physician involvement, the phage bank approach also fits less well into typical models of drug development and regulatory approval With regard to phage cocktails and their utility, it is important to recognize that different phages can display different pharmacokinetic as well as pharmacodynamic properties Within phage biology, though, polyphage refers to a combination of more than one phage type