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Phage approved in food, why not as a therapeutic?

Article  in  Expert Review of Anti-infective Therapy · January 2015

DOI: 10.1586/14787210.2015.990383 · Source: PubMed

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 Review

Phage approved in food, why

not as a therapeutic?
Expert Rev. Anti Infect. Ther. 13(1), 91–101 (2015)

Wessam A Sarhan and Bacterial resistance is not only restricted to human infections but is also a major problem in
Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by 82.201.218.20 on 12/09/14

Hassan ME Azzazy* food. With the marked decrease in produced antimicrobials, the world is now reassessing
bacteriophages. In 2006, ListShield
received the US FDA approval for using phage in food.
Novel Diagnostics and Therapeutics
Research Group, YJ-Science and Nevertheless, regulatory approval of phage-based therapeutics is still facing many challenges.
Technology Research Centre, School of This review highlights the use of bacteriophages as biocontrol agents in the food industry. It
Sciences and Engineering, The also focuses on the challenges still facing the regulatory approval of phage-based therapeutics
American University in Cairo, AUC
Avenue, SSE 1184, P.O. Box 74,
and the proposed approaches to overcome such challenges.
New Cairo, 11835, Egypt.
*Author for correspondence: KEYWORDS: bacterial resistance . bacteriophages . food biocontrol products . phage therapy
Tel.: +20 2 2615 2559
Fax: +20 2 2795 7565
hazzazy@aucegypt.edu In 1917, the term bacteriophage was first intro- resulted in several companies developing
duced by Felix d’Herelle who was also the first phage-based products for food, agriculture,
to test phage therapy. d’Herelle applied bacter- diagnostics and therapeutics (TABLE 1).
iophages against livestock infections and even
For personal use only.

tested them on himself. It was then recognized Bacteriophages: brilliant antibacterials

that the same phenomenon was described by Bacteriophages are the most abundant micro-
Twort in 1915 and by Hankin in 1896 in the organisms with an estimated number of
Ganges River against Vibrio cholerae [1–3]. It 1030–1032 phage particles. Humans are regu-
was in the 1920s, when d’Herelle used bacter- larly exposed to bacteriophages in water and
iophages for fighting different bacterial infec- unprocessed food. Moreover, phages are pres-
tions around the world that the discipline ent in the intestinal tract, saliva and in dental
known as ‘phage therapy’ was introduced. plaque. One milliliter of unpolluted water
Phage therapy rapidly developed globally and contains 2  108 phage particles [5,6].
was used as the major antibacterial in many Bacteriophages infect bacteria via two possi-
countries. Moreover, phage therapy also ble lifecycles: the lysogenic and the lytic. The
attracted the attention of many pharmaceutical infection begins with the adsorption of the
companies including E.R. Squibb & Sons bacteriophage onto the surface of the bacteria
(Princeton, NJ, USA), Eli Lilly (Indianapolis, then the viral genetic material is injected into
IN, USA) and Swan-Myers/Abbott laborato- the cytoplasm. Lytic bacteriophages take
ries, which produced commercial phage prepa- immediate control of the biochemical machin-
rations [4]. However, because phage properties ery of the host cell to make new virions and
were poorly understood and well purified and the growth cycle ends by killing the host
characterized preparations were lacking, phage cell [7]. In contrast, temperate bacteriophages
therapy resulted in variable outcomes and insert their genome into the chromosome of
many specialists questioned its efficacy. With the host cell and remain in the dormant state
the introduction of antibiotics in the 1940s, until the bacteria is exposed to certain stimuli,
bacteriophages were unable to stand against the which lead to initiation of the lytic cycle.
miracle antibiotics [4]. However, during the TABLE 2 illustrates attributes of bacteriophages
past two decades, which witnessed the continu- that make them attractive antibacterial thera-
ous rise in bacterial resistance together with peutics [8,9]. It is relatively easy and inexpensive
alarming decrease in the production of new to find new phages against bacterial resistance
antibiotics, the Western world is actively revis- compared with years and billions of dollars in
iting the world of bacteriophages in different case of antibiotic. Moreover, the ability of bac-
practical applications. An interest that has teriophages to kill pathogenic bacterial strains

informahealthcare.com 10.1586/14787210.2015.990383  2015 Informa UK Ltd ISSN 1478-7210 91

 Review Sarhan & Azzazy

Table 1. List of some bacteriophage-based resistant strain was reported in India which carried a gene
companies. called NDM1 (New Delhi metallo-b-lactamase). This was the
first report on spread of resistant strains in a community, as
Company Established Ref.
such incidences were restricted to hospitals [12]. The problem of
Omnilytics (USA) 1954 [98] bacterial resistance is further complicated with the marked
Exponential Biotherapies (USA) 1994 [99] decrease in introducing new antimicrobials. Major decrease in
antibiotic development occurred over the past 25 years. From
Biophage Pharma (Canada) 1995 [100]
2008 to 2012, only two antibiotics were under develop-
PhageTech (Canada) 1997 [101] ment [13,14], and from 2003 to 2007, only five antibiotics were
Intralytix (USA) 1998 [102]
underdevelopment compared with over 14 antibiotics back
between 1983 and 1987 [14].
Hexal Gentech (Germany) 1998 [103]
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Phage Biotech (Israel) 2000 [104] Applications of bacteriophages

Although the focus of Western countries on bacteriophages as
GangaGen 2000 [105]
(Bangalore, India; San Francisco, an antibacterial has almost faded with the introduction of anti-
CA; Ottawa, Canada) biotics, countries as the former Soviet Union with Georgia as
the epicenter continued their studies on bacteriophages to treat
PhageGen 2000 [106]
serious and chronic infections [14].
(Las Vegas, NV; previously Regma
Bio Technologies of London) In the 1980s, Smith and coworkers carried out several studies
to assess the effectiveness of phage therapeutics against E. coli
Phico Therapeutics (UK) 2000 [107]
infections [15–17]. Many other reports evaluated bacteriophage
Phage-Therapy (Tbilisi, Georgia) 2002 [108] therapeutic efficacy against different bacterial infections as E. coli
[18], Pseudomonas aeruginosa [19], Acinetobacter baumanii [20],
Ampliphi biosciences (Australia) 2002 [109]
Klebsiella pneumoniae [21], Vibrio vulnificus [22], as well as Salmo-
For personal use only.

Novolytics (UK) 2002 [110] nella [23] in animal models. Other studies revealed phage thera-
Technophage (Portugal) 2002 [111] peutic effectiveness against drug-resistant strains of bacteria, such
as vancomycin-resistant E. faecium [24] and methicillin-resistant
Enzobiotics/New Horizons 2003 [112]
S. aureus [25]. More remarkably, phages were able to reach intra-
Diagnostics (USA)
cellular bacteria, such as Mycobacterium tuberculosis [26].
Pherecydespharma (France) 2006 [113] Another important property of bacteriophages is their abil-
ity to disperse biofilms, although it was observed that antibi-
otic use at subminimal concentrations could end in induction
in situ through exponential growth of phage is significant for of biofilm formation [27,28]. Biofilms represent aggregation of
the treatment of chronic bacterial infections [8,10]. cells either eukaryotic or prokaryotic. Such cells are sur-
rounded by a matrix of extracellular polymeric substance. Bac-
Rediscovering bacteriophages teria found within biofilms possess increased resistance to
Since the discovery of penicillin, antibiotic production is esti- antibiotics and biocides, where it may require 1000-times
mated at US$25 billion per year. The widespread and some- more of such agents to eradicate biofilm bacteria compared
times inappropriate use of antibiotics has contributed to the with free-living bacteria [28]. Bacteriophages, however, have
increased incidence of bacterial resistance. The overuse of anti- proven ability to infect bacteria within biofilms [28]. Phage
biotics in animal feed has also contributed to antibiotic resis- replication within the bacterial cells results in localized ampli-
tance. Many of the 17 antibiotic classes are used in animal fication in phage numbers. Phages then spread within the bio-
feed. Although Europe has banned the use of antibiotics for film eradicating the bacteria producing the extracellular
promoting animal growth, it is still allowed in the USA [5]. polymeric substance. Moreover, some bacteriophages have the
In the USA, bacterial resistance costs the health-care system ability to express or carry depolymerizing enzymes that can
over US$20 billion every year, leading to over 8 million extra degrade extracellular polymeric substance [28]. Unfortunately,
hospital days. Moreover, the social costs are over US$35 billion the chance of isolating a natural phage that shows both advan-
every year. In the EU, multidrug-resistant bacteria result in the tages is low. Thus, researchers are now engineering phages to
death of nearly 25,000 patients annually and the associated express biofilm degrading enzymes [29]. Phage dispersing bio-
economical burdens are postulated to be nearly 1.5 billion films show strong potential not only on the therapeutic front
Euros annually [11]. but also at the industrial scale, where biofilms represent a con-
Several antibiotic-resistant bacteria have been identified tinuous source of bacteria on different surfaces. It was shown
including methicillin-resistant Staphylococcus aureus, also that phages and antibiotics are not mutually exclusive
vancomycin-resistant S. aureus [8] and vancomycin-resistant instead they could be synergistic, especially for managing bio-
Enterococcus faecium [11]. In 2010, a serious Escherichia coli- film infections [30,31].

92 Expert Rev. Anti Infect. Ther. 13(1), (2015)

 Challenges facing phage therapy Review

Table 2. Comparison between phages and antibiotics as antibacterials.

Bacteriophages Antibiotics
Very specific affecting only the targeted bacterial species with no Target both pathogenic microorganisms and normal microflora
disruption of normal flora therefore minimizing the possibility of which may lead to serious secondary infections
secondary infections
Autodosing through replication at the site of infection (repeated Metabolized and eliminated and do not accumulate at the site
administration may not be needed) of infection
No serious side effects have been described Multiple side effects, including allergies, intestinal disorders,
Minor side effects may be due to the liberation of endotoxins secondary infections, adverse effects on the kidney and the liver
from bacteria lysed in vivo by the phages
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Phage-resistant bacteria are not resistant to other phages having Resistance to antibiotics extends over targeted bacteria
a similar target range
Finding new phages against developed bacterial resistance can Developing a new antibiotic against antibiotic-
be achieved in days resistant bacteria is a very lengthy and expensive process
Ability to clear biofilms Limited ability of biofilm clearance

Many studies were done on the use of phage as a microbial Several reviews have addressed successful bacteriophage appli-
control in food, including meat, poultry, fish, fruits and vegeta- cations in human therapy [44–46] and in food either in the pre-
bles, both in the preharvest and postharvest (fresh and pack- harvest or postharvest stages [47].
aged foods) stages. Studies have been conducted on different
stages in the food production chain, including also livestock Phages approved as biocontrol agents in the food
decontamination, and sanitation of contact surfaces and equip- industry
For personal use only.

ment to control some of the most persistent foodborne patho- The market of food and beverage was valued at US$5.7 trillion
gens in poultry including Campylobacter jejuni [32] and worldwide in 2008 and is expected to reach US$7 trillion in
Salmonella typhimurium [33]. Phage was also applied in cattle 2014 [5]. Food poisoning and spoilage due to bacterial contam-
and sheep against E. coli O157:H7 [34]. However, more studies ination represent a major burden on health and food industry.
are required to enhance the in vivo biotherapeutic effects of The Centers for Disease Control estimated about 9.4 million
phages, including better understanding of the interactions cases of foodborne illnesses in addition to about 56,000 hospi-
between phage and bacteria in the gut and alimentary system talizations, and over 1350 deaths in the USA annually [48].
of live animals [35]. Moreover, microbial contamination is the major cause of food
Since the regulatory approval of ListShieldTM as the first spoilage, which results in loss of 25% of food produced each
phage-based product for control of Listeria in products of meat year [49].
and poultry, studies for phages targeting food (meat, fruits, Due to the increase in bacterial resistance, together with the
vegetables, processed ready to eat [RTE] food, cheese, pasteur- progress achieved in understanding phage biology and its appli-
ized milk, powdered infant formulas, etc.) in the postharvest cations, numerous companies worldwide are currently investing
stage have increased [36]. Guenther et al. (2009 and 2012) stud- in developing phage-based products as food biocontrol agents,
ied the effect of bacteriophages against Listeria monocytogenes decontamination, sanitation and diagnostics.
and S. typhimurium in RTE foods, respectively [37,38]. In 2005, the US Environmental Protection Agency approved
Kim et al. (2007) reported the use of a phage cocktail against AgriphageTM (OmniLytics Inc., Sandy, UT) [50] for treating
Cronobacter sakazakii in reconstituted infant formula [39]. bacterial spots in different crops. In 2006, the FDA approved
Anany et al. (2011) immobilized anionic heads of phages on ListShieldTM to be applied directly on RTE meat and poultry
cationic cellulose membranes leaving the tails to catch and kill to control L. monocytogenes [51]. Thus, bacteriophages were for
both L. monocytogenes and E. coli O157:H7 in RTE and raw the first time considered generally recognized as safe
meat [40]. (GRAS) [29]. TABLE 3 presents selected examples of using
Bacteriophages were also used as biocontrol agents in vegeta- approved phage products in food.
bles and fruits. Leverentz et al. (2001) used phage alone or in In 2007, FDA approved phage-based preparations produced
combination with bacteriocin to reduce Salmonella contamina- by Omnilytics to decontaminate live animals from E. coli and
tion in fresh cut fruits [41]. Whereas, Viazis et al. (2010) Salmonella [52]. Now, several products are commercially avail-
showed complete inactivation of E. coli O157:H7 strains in let- able including Finalyse spray against E. coli O157:H7 in cattle
tuce and spinach using a combination of phage mixture and and Armament against Salmonella in poultry [47].
trans-cinnamaldehyde oil [42]. In 2013, the combined use of The future of phage-based products in the food industry
bacteriophages with modified atmosphere packaging improved shows tremendous promise, especially with the number of com-
the effect of bacteriophages [43]. panies as well as research institutes actively engaged in phage

informahealthcare.com 93
 Review Sarhan & Azzazy

Table 3. FDA approved phage-based products for use in ready to eat food.
Product Regulatory approval Applications Ref.
ListShield
Intralytix, Inc. US FDA (2006) and USDA for direct application – Ready to eat food: salami, sausage, [114]
USA onto foods (21 CFR 172.785.) EPA (EPA basterami, etc.
registration 74234-1) – Sea food
– Food contact surfaces and enviroments
EcoShield Intralytix, Inc. FDA (2011) cleared as ‘Food Contact Notification’ – Red meat parts and trim intended to be ground [115]
USA or FCN, (FCN No. 1018). FSIS Directive
7120.1 (safe and suitable antimicrobial)
SalmoFresh Intralytix, Inc. FDA (GRAS Notice No. GRN 000435), – Poultry [116]
USA FSIS Directive the Star K-certified Kosher and – Fish and shellfish
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IFANCA-certified Halal product. – Fresh and processed fruits and vegetables

OMRI-listed suitable in the production of organic
foods
LISTEX
Micreos EBI In 2006 approved by the FDA as GRAS, and by – Meat [117]
Food Safety the USDA in 2007 and by the EFSA, Health – Ready to eat meat
Netherlands Canada, BAG (Switzerland) and FSANZ (Food – Fish
Standards Australia New Zealand) – Cheese
Agriphage Omnilytics EPA 2005 for use in agriculture – In agriculture on fruits and vegetables [118]
USA

research. However, this is not the case for phage therapy in anaphylactic shocks. Phages are recognized as foreign antigens
humans, where a number of challenges still impede its by mammalian hosts [54].
For personal use only.

approval. It was initially reported that the reticulo-endothelial system

was responsible for the decrease in the concentrations of bacter-
Phages not approved as therapeutics; challenges still in iophages in the blood of mice [55]. Decades later it was proven
the way that the liver played the major role in phage elimination with
Despite the long history of phage therapy in addition to its over 99% of the phages present in the circulatory system were
safety record, phages are still not approved by the FDA to be phagocytosed by the liver [56]. Such elimination of phage by
used as therapeutics. Back in the 1970s, 1980s and 1990s, the the reticulo-endothelial system, coinciding with lack of infor-
FDA revised the usage of phage preparations as therapeutics in mation on phage pharmacokinetics, has resulted in majority of
human. At the time, phage phiX174 was applied via intrave- phage applications being administered orally [57]. Consequently,
nous administration to patients with immunodeficiency such as despite the advantageous therapeutic effects of phages on sys-
those infected with HIV. In the 1970s, after the inclusion of temic diseases, most studies to date were concerned with the
phages in several vaccines, the FDA performed a safety review treatment of non-systemic diseases, such as gastrointestinal, ear
on phages and concluded that bacteriophages are safe and and wound infections [58]. Merril et al. [59] serially passed
allowed the continued usage of the vaccines [7]. phages in animals to isolate mutants that were capable of stay-
In 2008, the first phage Phase I clinical trial was approved ing longer in circulation and allowing enhanced systemic
by the FDA. The study evaluated the use of a phage prepara- administration [59].
tion of eight phages against venous leg ulcers. The trial verified In addition to the interaction with the innate immune sys-
the safety of the phage cocktail preparation [53]. tem, bacteriophages also stimulate the adaptive immune system
Despite the documented safety of bacteriophages, however, and consequently the production of antibodies [60]. Less anti-
regulatory bodies have not approved its use as a therapeutic, genic bacteriophages may be developed through phage display-
although recently approving it in food. Such approval confirms ing certain peptide ligands; Sokoloff and coworkers injected a
that phage is safe for human consumption and thus represents T7-phage peptide display library in rats, and observed that
a major step towards its approval as a therapeutic. Challenges phage displaying peptides with arginine or carboxy-terminal
that currently hinder regulatory approval of phage as human lysine residues protected against the complement-mediated
therapeutics as well as possible approaches to overcome them inactivation of the bacteriophage by binding C-reactive protein,
will be discussed (FIGURE 1). whereas human serum phages that displayed C-terminal tyro-
sine residues were resistant to inactivation [61].
Immunogenicity Within this context, Kim et al. were the first to introduce
Phage immunogenicity is a major challenge facing phage ther- PEGylation to bacteriophages to increase bacteriophages sur-
apy, mainly because of its effects on phage pharmacokinetics vival and efficacy [62]. Consistent with this, Molenaar et al.
and also because of the possible side effects, such as have shown that the incorporation of targeting ligands

94 Expert Rev. Anti Infect. Ther. 13(1), (2015)

 Challenges facing phage therapy Review

increased the efficient delivery and specificity of the

M13 phage to liver Kupffer and parenchymal cells [63]. Regulatory
Manufacturing Pharmacokinetics
S-radiolabeled M13 bacteriophage was chemically modified by approval
conjugation of either succinate groups or galactose to the phage
coat protein to facilitate the uptake of the phages by scavenger
receptors and hepatic receptors, respectively. Although the main Challenges
Bacterial Narrow host
purpose of this study was to increase efficiency of in vivo pan- facing phage
resistance range
therapy
ning of phage libraries, the same principle could be used for
targeting other organs for therapeutic purposes [63].
Systemic side
Pharmacokinetics Immunogenicity Patentability
effects
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The pharmacokinetic behavior of phages was only weakly

addressed. Efficient pharmacokinetic data are required to sup-
port phage delivery via the bloodstream to achieve systemic Figure 1. Challenges facing phage therapy.
effects. Phages replicate exponentially, interact and show differ-
ent phenomena completely different from the chemical kinetics
of conventional drugs [64]. Consequently, studying the pharma- bacterial treatment will generate endotoxins and superantigens
cokinetics of bacteriophages as antibacterial therapeutics necessi- that may be released during the massive and fast destruction of
tates knowledge of the infected host, the bacteriophage and the bacteria in vivo [58]. Although this possibility may be low, engi-
infecting bacteria as well as their complex interactions. Interest- neered phages that are either lysis deficient or non-replicative
ingly, through the process of infection and phage therapy, the have been developed. For example, filamentous phages were
growth of bacteria would lead to exponential growth of phages, engineered to express certain toxic genes in E. coli leading to
unlike the conventional use of antibiotics [64]. Along this line, bacterial toxicity but not their lysis, thus overcoming the prob-
Dubos and coworkers treated mice that were infected intracere- lem of endotoxins [69]. Non-lytic and non-replicative phages
For personal use only.

brally with Shigella dysenteriae by administering phage into the were also designed against P. aeruginosa [70]. Interestingly, in
peritoneal cavity [65]. Similar studies by Smith and Huggins both the studies, it was observed that the non-lytic phages
demonstrated that mice infected either intracerebrally or intra- increased the survival of treated infected mice compared with
muscularly with E. coli were rescued with phages via intramus- lytic phages, which may have been caused by reduced
cular administration [15]. In both the studies, the levels of the inflammation.
bacteriophages were highest in the infected tissues but then It is also important to ensure that bacteriophages for ther-
decreased as the bacterial levels decreased. On the other hand, apy do not carry gene sequences that possess significant simi-
phage levels in the blood of the control animals were in accor- larity with antibiotic-resistance genes, genes for other bacterial
dance with the phage dilution in the total blood of the animal. virulence factors and genes for phage-encoded toxins. More-
These data were used as the basis for mathematical models pre- over, phages should be also incapable of generalized transduc-
dicting the kinetic behavior of phages [64,66]. Several parameters tion [47]. A number of quality assurance and quality control
should be considered to achieve effective phage therapeutics. measures to consider are described in Merabishvili et al.
These include the route and timing of administration and the (2009) [71].
required dose [64].
Narrow host range
Systemic side effects Each phage strain infects only one type of bacteria. The down-
Lytic phages are the only phages that are used in therapy. First side of such narrow host range is the limited applicability in
because they are more potent than the temperate phages, and cases where the identification of the specific bacterial host can-
second because temperate phages have the risk of transferring not be achieved [7]. However, with the tremendous develop-
fragments of the DNA of the host to other bacterial species, ments of rapid diagnostics, identifying the specific bacterial
thus posing the risk of producing virulent strains if such frag- host is easily achieved. In most situations, however, bacterio-
ments were toxin encoding or antibiotic-resistant genes. phage cocktails are used to broaden the host range and increase
No reports have been recorded of serious complications the overall therapeutic efficiency. It is unlikely, however, to
accompanying phage therapy. Moreover, humans are in contin- achieve 100% therapeutic efficiency [7,58]. Thus, the use of sin-
uous exposure to bacteriophages, as they are so common in the gle phages versus cocktails of phages has to be further studied
environment. For example, unpolluted water contains ca. 2  and optimized as both regimens may lead to the development
108 (phage/ml), and phages are continuously consumed in of resistance [72]. Nevertheless, as will be discussed later,
foods [44]. approval of phage cocktails is problematic, thus other methods
Safety of bacteriophages was proved through different studies have also to be developed. Using broad host spectrum bacterio-
where bacteriophage did not cause adverse effects in phages, such as Listeria P100 or S. aureus phi812 was pro-
humans [67,68]. However, there are concerns that any lytic posed [73,74]. Also, broad host range could be achieved via

informahealthcare.com 95
 Review Sarhan & Azzazy

grafting the g3p phage protein of one filamentous bacterio- Manufacturing; (safety & efficacy)
phage to another [75,76]. Such an approach, however, although In the past, and in Russia until now, phages have been admin-
would broaden the host range of the engineered bacteriophage istered to humans: orally, either in tablets or liquid formula-
and would facilitate its patentability, may also complicate its tions (105 to 1011 PFU/dose); locally to the skin, ear or eye in
regulatory approval. Moreover, safety of such engineered the form of creams, tampons and rinses; rectally; as aerosols; as
phages has not been thoroughly studied, especially that such intrapleural injections; and intravenously [45]. Still, there are
engineered phages may increase the probability of phage- two critical factors that need to be standardized during bacteri-
mediated transfer of genetic sequences among bacterial ophage manufacturing, which are safety and efficacy. Inability
species [77]. to optimize these factors may have been responsible for the
Developing clinical diagnostics to allow rapid identification recorded negative results of phage therapy. Efficacy of the
of the infective bacterial pathogen as well as their phage suscep- phage preparation reflects the presence of sufficient virulent
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tibility may be necessary to allow the use of the required phage phages, especially against the target bacteria [83]. Poor viability
instead of phage cocktails. These phage-based diagnostics and/or stability of phage preparations may result from
should be fast and reliable [78]. improper manufacturing techniques. Therefore, in some early
Along this line, Loessner and coworkers [78] developed lucif- commercial phage preparations, mercurials and oxidizing agents
erase reporter bacteriophages for the sensitive and fast detection as well as heat treatments were used to ensure bacterial sterility.
of viable cells of Listeria. In addition, diagnostic tests that uti- Nevertheless, such treatments may result in ineffective prepara-
lized phage enzyme lysins have been developed allowing rapid tions due to phage inactivation. However, advanced
bacterial identification and have resulted in commercialized manufacturing techniques are now utilized for phage purifica-
products (Enzobiotics; Columbia, MD, USA) [9]. tion. Moreover, determination of the phage titer and viability
is essential [83].
Bacterial resistance Safety on the other hand is a very important parameter, as
Bacteriophages are the most abundant and diverse microorgan- issues related to the presence of exotoxins and pyrogens released
isms on the planet. Such diversity is due to the continuous during bacterial lysis by bacteriophages have always raised seri-
For personal use only.

adaptation to different pressures. Bacteriophages have evolved ous safety concerns. Consequently, bacteriophage manufacturing
different mechanisms that enable it to avoid, withstand and get for clinical use must be optimized to eliminate bacterial exotox-
over different mechanisms of bacterial resistance [79]. It was ins and pyrogens [60]. Early therapeutic phage preparations suf-
found that in monoculture studies performed in vitro, phage fered from inefficient purification resulting in crude lysates of
resistance can evolve within hours or days [58]. However, it was the host bacteria in the preparation [84]. Efficient purification
observed that bacteria develop phage resistance in vitro more can be achieved by ammonium sulfate precipitation and also by
rapidly than in vivo [80]. Thus, the correlation between the rate CsCl gradient centrifugation [84]. Ultrafiltration and two-step
of resistance development in vivo and in vitro is an area that chromatography were also used [84]. However, the purification
requires further study. procedures have witnessed considerable evolution in the past few
The exposure of a certain bacterial strain to a single bacterio- years. Recently, Kramberger and coworkers [85] utilized methac-
phage is suggested to aid in the emergence of phage-resistant rylate monoliths columns in S. aureus phage purification from a
strains of the bacteria. On the other hand, several studies report lysate of bacterial cells. This method resulted in recovery of
that phage cocktails help control or delay the evolution of 60% of viable phages in a single purification step [85]. In this
phage-resistant strains [72,81,82]. Reduction of appearance of context, Gill and Haymen, published an interesting review
resistant bacterial strains can be achieved using new techniques, exploring the different available methods for bacteriophage puri-
such as using combinations of bacteriophages with other anti- fication as well as the different levels of purification required for
microbials (such as antibiotics or antimicrobial herbal extracts), different applications together with the current protocols utilized
cycling bacteria through mixtures of different phages and tar- in the commercial production of different products of phage
geting phage-resistance mechanism by genetically engineered therapy [83]. Standard purification techniques should be devel-
bacteriophages [58]. oped to achieve clinical grade bacteriophage preparations.
In this context, additional studies are needed to identify
phage receptors and understanding the interaction of bacterio- Regulatory approval
phages with the receptors on the host surface. Until now, Despite the successful application of phage therapy for decades
except for E. coli, very few phage receptors have been identi- in some eastern countries such as Poland and the former Soviet
fied [79]. Moreover, the evolution of phages in response to the republics, especially Georgia and Russia [86], their clinical data
mutations in the host surface receptors needs to be investi- failed to gain regulatory approval because such data were not
gated. Finally, the environment in which phage-resistance developed under the regulatory authorities frameworks.
mechanisms are studied need to be translated from a closed A major hurdle in the way of phage becoming part of the
laboratory environment with single phage at a time to a more mainstream medicine is the absence of well-defined guidelines
real complex environment containing multiple anti-phage for regulatory approval of phage. Instead, phage is regulated
barriers [79]. according to existing guidelines developed for typical

96 Expert Rev. Anti Infect. Ther. 13(1), (2015)

 Challenges facing phage therapy Review

antibacterials [7], meaning that every component of the thera- only genetically engineered bacteriophages can meet the patent
peutic cocktail of phages must undergo individual clinical trials regulations. Several patents, however, have been granted by the
and that the composition of the approved phage cocktail can- US patent office, including a patent for reduction of sepsis
not be changed without re-approval. Such regulations do not with a pharmaceutical preparation including bacteriophages, as
take into consideration the differences between phages and well as other patents approved for using phage in the food sec-
antibiotics. Fortunately, there exists a regulatory framework tor. A detailed discussion of patent issues related to therapeutic
that could be applied for phage therapy, which is that of the use of phage is described elsewhere [93–95].
influenza live-virus vaccine. Such a vaccine is formulated from
a cocktail of three or four influenza strains that the FDA has Expert review
approved to be reformulated annually according to the circulat- The regulatory approval of phage consumption in food pro-
ing flu strain [7]. vides a validation for its safety for humans. Thus, the main
Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by 82.201.218.20 on 12/09/14

However, with the recent approval of different phage prepara- hurdles still facing the regulatory approval of phage as thera-
tions in food and agriculture together with the approval of some peutics include the need for more clinical evidence for its thera-
clinical trials, the situation may be improving. Recently some peutic efficacy as well as new or modified regulatory guidelines.
Phase I studies have been carried out and published [87], whereas Such clinical evidence must be supported by detailed data on
others are being conducted but to date have not been pub- phage immunogenicity, pharmacokinetics and formulations.’
lished [53]. In 2009, Biocontrol Ltd Company (Nottingham, Some approaches for overcoming the challenges that hinder
UK) has completed randomized, double-blind, placebo-con- the approval of such an important therapeutic are discussed
trolled, fully regulated Phase II clinical trial for phage therapy below.
against Pseudomonas infections in the western world [54]. Another
. Bacteriophages act as exogenous antigens inducing a humoral
FDA-approved Phase I clinical trial in 2008 used phage to target
reaction due to the presence of immunogenic epitopes in
P. aeruginosa, E. coli, and S. aureus in venous ulcers [87].
phage proteins [64]. Masking or modifying these recognition
An important approach that should be considered by the
epitopes is needed to improve phage blood circulation and
regulatory authorities is regulating phages for the ‘pharmacy
For personal use only.

decrease immunogenic response. Chemical modification

approach’. The approach allows phage cocktails to be custom
through PEGylation of the bacteriophages and/or addition of
made by licensed hospital pharmacists from current Good
biocompatible moieties can be used to improve phage phar-
Manufacturing Practice phage preparations instead of all hospi-
macokinetics and decrease its immunogenicity [61,63].
tals using the same preparations [7].
. Phage pharmacokinetics. The basic kinetic principles of self-
In Europe, and pending resolution of regulatory issues, phage
replicating phages have been recently addressed in a number
therapy is used under the supervision and responsibility of medi-
of important studies [7,64] which will allow better experimen-
cal ethical committees within the approval of the World Medical
tal design and data interpretation. There is still a need to
Association Declaration of Helsinki [88,89]. This is only a tempo-
develop kinetic models describing the density dependent bac-
rary solution that will not result in efficient introduction of
teria–phage interactions [8].
phage therapy into the western or the rest of the world [53].
. Optimization of the formulation and long-term stability. A
small-scale quality controlled production of defined bacterio-
Patentability
phage cocktail for use in human was developed and resulted
Recently, a patent by Heo and coworkers was granted and was
in approved clinical trials [71].
based on bacteriophage MPK6, a novel bacteriophage belong-
. The approval of phage in food (TABLE 1) should motivate
ing to the order Caudovirales, and its pharmaceutical composi-
researchers and the pharmaceutical industry to pursue more
tion for treating P. aeruginosa [90,91]. The issue of phage
clinical evidence for its therapeutic efficacy and to work with
patentability resembles that of patenting monoclonal antibod-
the regulatory agencies to develop new guidelines for phage
ies, where in both cases a wide array of potential targets exist
therapy. This is now important with the alarming rise of bac-
and the methodology for their discovery is common knowl-
terial resistance and the low number of new antibiotics
edge. Individual bacteriophages are patentable where they can
introduced [86].
be put under the Budapest treaty in an approved collection [92].
However, in both the cases, there are always other agents
(either antibodies or phages) that can be discovered, isolated Five-year view
and optimized to formulate even more efficient bacteriophage The application of bacteriophages in different food sectors is
preparations [53]. The European patent laws allow patenting of expected to increase supported by the regulatory approval of
known phages provided that the use of these phages has not different phage products (TABLE 1).
been disclosed [93]. Moreover, the new phage formula can be With the alarming increase in bacterial resistance together
patented [53]. The European patent office necessitates a certain with the decreased number of new antibiotics, the current regu-
technical intervention for isolating phage from its natural envi- latory guidelines may be modified in favor of phage therapy.
ronment, as well as proper characterization of the isolated Developing specific and simple tests that allow rapid identifi-
phage. For the US Patent and Trademark Office, however, cation of the causative bacterial strain as well as their phage

informahealthcare.com 97
 Review Sarhan & Azzazy

susceptibility is one of the major areas that will aid in phage Financial & competing interests disclosure
adoption as therapeutics. Along this line, nano-based tests will W Sarhan and H Azzazy are authors of a patent application describing
be one of the fast developing areas in the upcoming years [96]. the use of nanofibers and phage for wound healing. The authors have no
It is expected that stable phage formulations alone or with other relevant affiliations or financial involvement with any organization
other antibiotics or natural antibacterials may be developed or entity with a financial interest in or financial conflict with the subject
with the aid of novel nanomaterials [97]. matter or materials discussed in the manuscript apart from those disclosed.

Key issues
. In 2011, the WHO has made the call ‘No action today, no cure tomorrow’ raising the need for immediate solutions against the serious
problem of bacterial resistance.
Expert Review of Anti-infective Therapy Downloaded from informahealthcare.com by 82.201.218.20 on 12/09/14

. Phage holds a substantial antibacterial potential as a therapeutic and a bio-control agent in food.
. In 2006, US FDA approved the first phage product to be applied directly to ready-to-eat meat.
. Different challenges are still in the way for approval of phage therapy, such as: immunogenicity, pharmacokinetics, systemic side
effects, narrow host range, bacterial resistance, manufacturing safety and efficacy, regulatory approval and patentability.
. The regulatory approval for phage consumption in food should motivate the pharmaceutical industry and scientific community for
developing bacteriophage therapeutics.

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