Phage Cocktails and the Future of Phage Therapy

Benjamin K Chan; Stephen T Abedon; Catherine Loc-Carrillo

Future Microbiol. 2013;8(6):769-783.

Abstract and Introduction

Abstract

Viruses of bacteria, known as bacteriophages or phages, were discovered nearly 100 years ago. Their potential as antibacterial agents was appreciated almost immediately, with the first 'phage therapy' trials predating Fleming's discovery of penicillin by approximately a decade. In this review, we consider phage therapy that can be used for treating bacterial infections in humans, domestic animals and even biocontrol in foods. Following an overview of the topic, we explore the common practice – both experimental and, in certain regions of the world, clinical – of mixing therapeutic phages into cocktails consisting of multiple virus types. We conclude with a discussion of the commercial and medical context of phage cocktails as therapeutic agents. In comparing off-the-shelf versus custom approaches, we consider the merits of a middle ground, which we deem 'modifiable'. Finally, we explore a regulatory framework for such an approach based on an influenza vaccine model.

Introduction

Bacteriophages, or simply phages, are viruses with an ability to infect and, in many cases, kill bacterial cells. As with most viruses, these infections begin with virion binding to specific cell-surface receptors, which is then followed by intracellular replication. Over 90% of phages have tailed icosahedral heads that, after inserting their nucleic acid into the bacterial cell, terminate their infections by lysing the cells they have infected. Depending on the life cycle of the phage, this lysis can occur either soon after the initiation of infection (lytic cycle) or instead following lengthy periods of delay (lysogenic cycle).^[1] In addition to permanently shutting down bacterial metabolism, lysis also releases phage progeny into the surrounding environment, allowing them to infect similar bacteria found nearby.

Similarly to antibacterial agents such as antiseptics and antibiotics, a crucial aspect of phage functioning as biological antibacterials is their potential to be applied directly to living tissues without causing harm, that is, they demonstrate selective toxicity. Though not always emphasized, especially historically,^[2] an important component of selective toxicity is an ability to avoid harming the often useful normal microbiota that are associated with mammalian bodies.^[3,4] Therefore, displaying a narrow spectrum of activity can be a useful property for an antibiotic or equivalent antibacterial. Furthermore, the host range of phages, as equivalent to their spectrum of activity, tends to be relatively narrow, often consisting of only a subset of strains making up a single bacterial species.^[5] This same characteristic can be limiting, however, in terms of the ability of specific phage products to impact bacterial infections.

Using phages to treat bacterial infections (commonly termed phage therapy) dates back to the early 1900s, after their codiscovery by Frederick Twort^[6] and Felix d'Hérelle.^[7] D'Hérelle in particular used phage suspensions to treat infections such as dysentery, which at the time had no other consistently effective treatment. His success led to a period of widespread enthusiasm for the phage therapy of humans.^[8] Although eclipsed in much of western medicine upon the advent of antibiotics, the use of phages as the treatment of choice for bacterial infections has persisted in various regions of the world.^[9] This includes, most notably, the former Soviet Republic of Georgia, where phages are often used as the standard of care for bacterial infections. In addition, there is the phage therapy center of Wrocław, Poland, where phages are used to treat especially chronic bacterial infections that are proven to be resistant to antibiotic treatment.^[10]

As a pioneering medical innovation, phage therapy came to be implemented well before the underlying science was even rudimentarily understood. The use of phages as therapeutic agents consequently had many problems stemming especially from a profound lack of understanding of phage biology.^[11] In particular, as with any antimicrobial therapy, it is crucial to employ agents that have some potential, first in vitro and then in vivo, to serve as effective antagonists to target organisms, and that are also safe in their application. These and other critical characteristics, which we now know should be required of phages before they can be considered as suitable candidates for therapeutic use, were underappreciated in the early days of phage therapy. In addition to the need for therapeutic phages to be both active and sufficiently antagonistic against the bacteria being targeted, it is imperative to employ phages that are incapable of infecting bacteria lysogenically and that neither encode nor are capable of transducing bacterial virulence factor genes. Also worth mentioning is the necessity for phage preparations to be adequately purified (e.g., to remove bacterial debris). Such purification should be substantial (e.g., to remove most bacterial components, including endotoxins), particularly when phages are to be delivered directly to an animal's systemic circulation.^[12,13]

The primary criticism of phage therapy is generally that a paucity of modern, double-blinded, Phase III (efficacy) clinical trials in humans have been undertaken and, therefore, a substantial uncertainty exists within western medical practice regarding the potential for phage therapy to cure disease. A number of recent reviews have investigated the question of phage therapy efficacy explicitly in humans.^[8–10,14] For the most part, phage therapy has not yet met the 'gold standard' of double-blind efficacy determination. The lack of such studies, however, is primarily a consequence of these types of studies not being undertaken owing to a relative lack of funding for such endeavors rather than because they have been undertaken and failed to show evidence of efficacy. Phage therapy consequently exists as an older technology that seems to have shown great promise, both in terms of commercial use both for human treatment (e.g., Pyophage and Intestiphage sold in the former Soviet Union) and in the form of biocontrol products (as sold by OmniLytics [UT, USA] and Micreos Food Safety [The Netherlands]). Nonetheless, at present most phage products have not been subjected to sufficiently rigorous analysis, particularly in terms of the clinical treatment of humans. In this article we thus consider the use of phages as antibacterial agents as a maturing technology.

We first focus on more recent research, investigating the use of phage cocktails to broaden the spectrum of activity of therapeutic phage formulations. We then consider commercial development and related issues of regulation, especially of polyphage cocktail use as antibacterial drugs. We do not present information on monophage therapy since this involves using single-phage preparations and previous reviews have covered this area extensively.^[15,16] Overall, we are fairly confident of the medical potential of phages to treat antibiotic-resistant bacterial infections given the Georgian, Polish and other experiences. We are less certain, however, of the potential of phage therapy to become well integrated into most western models of drug development, regulation and clinical implementation.

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Next Section

Abstract and Introduction
Phages & Phage Therapy Basics
Polyphage Therapy
Integrating Phages & Phage Cocktails Into Clinical Medicine
Conclusion
Future Perspective
References
Sidebar

Table 1. Recent publications investigating phage cocktails for bacterial treatment control.
Target	Entity	Characteristic	Dosing	Phages^†	Reduction^‡	Comments	Ref.
Pseudomonas aeruginosa	Murine	Lung	in.	m × 1, p × 1	10^0.5	Bioluminescent quantification	[89]
Klebsiella pneumoniae	Murine	Bacteremia	ip.	m × 1, s × 2	–	100% recovery with 3 h delay from lethal dose	[40]
P. aeruginosa	Wax moth	–	–	m × 3, p × 1	–	Doubling of lifespan	[31]
Vibrio cholerae	Rabbit	Enteric	Oral	u × 5	10⁵	6 h delay	[41]
Escherichia coli	Murine	Enteric	Oral	m × 1, p × 1, s × 1		Biofilm reduction	[34]
E. coli	Food	–	–	u × 3	10¹	Relative to bacterial growth	[90]
Listeria monocytogenes	Food	–	–	u × 3	10^1.7	Relative to bacterial growth	[90]
Salmonella enterica ser. Typhimurium	Pig	Skin	–	m × 2, s × 2	10¹	Pig skin model	[91]
Enterococcus faecalis^V	Surfaces	–	–	m × 1, s × 1 or s × 1, s × 1	<10^1.5	Reductions on glass, cotton and polyester	[92]
E. coli	Leafy greens	–	–	u × 8	10⁵	E. coli O157:H7 strain	[39]
E. coli	Surfaces	–	–	u × 8	10³	E. coli O157:H7 strain	[84]
Campylobacter jejuni	Poultry	Colonization	Oral	m × 3	10²	Reduction in feces greater with phage delivered in feed	[87]
P. aeruginosa	Catheter	Biofilm	–	u × 3	<10²	Relative to bacterial growth	[81]
P. aeruginosa	Dog	Otitis	Topical	u × 6	<10²	Chronic disease, 24 h post-phage treatment	[93]
Clostridium perfringens	Poultry	Necrotic enteritis	Oral	u × 5 (s or m)	–	92% reduced mortality with phage treatment	[78]
E. coli	Poultry	Colibacillosis	Oral	m × 2, s × 1	–	Several-fold reduction in flock mortality due to natural infections	[42]
E. coli	Cattle	Colonization	Oral	m × 1, s × 1	–	Significant reduction in ex vivo but not in in vivo model	[94]
S. enterica ser. Typhimurium	Pigs	Colonization	Oral	m × 6, s × 9	10^1.4	Microencapsulation of phages for delivery	[88]
E. coli	Sheep	Colonization	Oral	m × 3	–	E. coli O157:H7 strain; reduction to zero in 60 versus 0% without phages	[85]
None	Murine	None	Oral	m × ?	NA	No adverse effects	[95]
K. pneumoniae	Murine	Burn wound	ip.	u × 5	–	Survival increase from 6 to 94%	[86]
P. aeruginosa	Murine	Burn wound	ip.	u × 5	–	No significant impact on survival	[96]
E. coli	Cattle	Colonization	Oral	m × 4	–	Marginally positive reductions	[97]
P. aeruginosa	Human	Otitis	Topical	u × 6	10¹	Phase VII clinical trial; indication of efficacy with no adverse effects	[44]
E. coli	Food	–	–	m × 3	–	Reductions up to 100%	[79]
Salmonella enteritidis ser. Enteritidis	Poultry	Colonization	Spray	u × 3	10^1.7	Phage treatment prior to bacterial inoculation	[98]
S. enteritidis ser. Enteritidis	Poultry	Colonization	Oral	u × 4 or u × 45	–	Substantial early reductions in colonization (45–100%) but less later (15%)	[99]
P. aeruginosa	Murine	Burn wound	im., ip. or sc.	u × 3	–	12% mortality with phage treatment (ip.) and 94% without	[43]

Entries are listed in decreasing date and then author alphabetized order.
^†Lettering indicates different phage types – Myoviridae (contractile tails), Podovirdae (short tails), Siphoviridae (long noncontractile tails) and uncertain – with one letter for each phage used in a cocktail. Standard practice is to propagate cocktails as individual phage stocks that are then mixed together.
^‡Indicated is maximum average reduction seen.
Enterococcus faecalis ^V: Vancomycin-resistant Enterococcus faecalis; im.: Intramuscular; in.: Intranasal; ip.: Intraperitoneal; m: Myoviridae; NA: Not applicable; p: Podovirdae; s: Siphoviridae; sc.: Subcutaneous; ser.: Serovar; u: Uncertain.

Table 2. Models of formulation for antibacterial treatment.
Type of formulation	Approach	Antibacterial types used per formulation	Personalized medicine	Prior to use characterization of infection^†	Breadth of spectrum of activity	Flexibility (to bacterial resistance)^‡	Flexibility (in spectrum of activity)^§
Phage bank (monophage)	Sur-mesure	1	+++	+++	+	+++	+++
Personalized cocktail	Sur-mesure	>1	+++	+++	++	+++	+++
Cocktail bank	Sur-mesure via prêt-à-porter	>1	++	++	+++	++	+
Single cocktail	Prêt-à-porter	>1 or >>1	–	+	+++	–^¶	–
Typical antibiotic	Prêt-à-porter	1	–	+	++++	–	–
Narrow-spectrum antibacterial^#	Prêt-à-porter	1	+	≥+	≥+	–	–
Single cocktail	Prêt-à-porter but modifiable	>1 or >>1	+	+	+++	+^††	++

^†Degree to which etiologies must be identified to achieve reasonable likelihood of treatment success.
^‡Potential to respond, in a clinic, to treatment failures resulting from infection resistance to a given phage formulation.
^§Potential for modification of formulation used for treatment over subyear time scales.
^¶Should phages prove able to adapt in vivo to resistant bacteria, then this designation may be modified towards increased flexibility; see [28] for discussion of this potential.
^#Such as monoclonal antibodies [100], enzymes [101] or bacteriocins [102]; proposed characteristics assume that the antibacterial diversity of such agents is small relative to that of phages.
^††This designation refers to an inability to respond to resistance in specific patients when using a fixed cocktail, but nonetheless that such patients may be subsequently treated given sufficiently rapid updating of otherwise modifiable cocktail formulations by manufacturers.

Box 1. Checklist towards successful phage therapy experimental development.
*Relevant for all approaches* Phages should be lytic and nontemperate (unable to display lysogenic infections) Phages should be able to lyse representative strains of target bacteria [34,78,79] Phage preparations should be purified to a level that is appropriate for the model used [80] Models should be sufficiently representative of relevant 'real-world' aspects [81] Efforts should be made to test surviving bacteria for phage resistance, retention of pathogenicity and genetic relationship to originally targeted bacteria [10,31,82] Combinations of phages should be tested towards cocktail optimization [31,83] Efforts should be made to avoid phage contact with bacteria during bacteria enumeration **Relevant for in vitro models Phage purification by filtration can be adequate for these studies Peak phage titers should reflect what can be achieved in the vicinity of target bacteria during the 'real-life' scenario being modeled [39,84] Relevant for in vivo and/or in situ models** Highly purified phage preparations are desirable to avoid formula-associated in vivo immune responses as well as experimental results other than those stemming from phage-mediated action For topical application, filtered phage lysates may suffice, although a phage-minus mock formulation may be necessary to distinguish phage- from nonphage-mediated effects Timing of administration should be considered to avoid especially unrealistic positive results that can stem from premature phage application following bacterial challenge [85] Phage persistence ability in vivo, in the absence of target bacteria, should be determined [34,86] In vivo toxicity testing of phage formulation should be performed Various in vivo routes of administration should be evaluated for effectiveness [87] Bacterial counts should be sampled from sites where bacteria can contribute to disease [88]
The terms in vitro , in vivo and in situ refer to experimentation using laboratory cultures, model organisms (e.g., animal models) and real-world circumstances (e.g., in the clinic, farm or kitchen), respectively. References cited provide helpful illustrations.