Literature Review

Phage approved in food, why not as a therapeutic?

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DOI: 10.1586/14787210.2015.990383 · Source: PubMed
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Abstract
Bacterial resistance is not only restricted to human infections but is also a major problem in 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. Nevertheless, regulatory approval of phage-based therapeutics is still facing many challenges. This review highlights the use of bacteriophages as biocontrol agents in the food industry. It also focuses on the challenges still facing the regulatory approval of phage-based therapeutics and the proposed approaches to overcome such challenges.
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Phage approved in food, why
not as a therapeutic?
Expert Rev. Anti Infect. Ther. 13(1), 91–101 (2015)
Wessam A Sarhan and
Hassan ME Azzazy*
Novel Diagnostics and Therapeutics
Research Group, YJ-Science and
Technology Research Centre, School of
Sciences and Engineering, The
American University in Cairo, AUC
Avenue, SSE 1184, P.O. Box 74,
New Cairo, 11835, Egypt.
*Author for correspondence:
Tel.: +20 2 2615 2559
Fax: +20 2 2795 7565
hazzazy@aucegypt.edu
Bacterial resistance is not only restricted to human infections but is also a major problem in
food. With the marked decrease in produced antimicrobials, the world is now reassessing
bacteriophages. In 2006, ListShieldreceived the US FDA approval for using phage in food.
Nevertheless, regulatory approval of phage-based therapeutics is still facing many challenges.
This review highlights the use of bacteriophages as biocontrol agents in the food industry. It
also focuses on the challenges still facing the regulatory approval of phage-based therapeutics
and the proposed approaches to overcome such challenges.
KEYWORDS:bacterial resistance .bacteriophages .food biocontrol products .phage therapy
In 1917, the term bacteriophage was first intro-
duced by Felix dHerelle who was also the first
to test phage therapy. dHerelle applied bacter-
iophages against livestock infections and even
tested them on himself. It was then recognized
that the same phenomenon was described by
Twort in 1915 and by Hankin in 1896 in the
Ganges River against Vibrio cholerae [13].It
was in the 1920s, when dHerelle used bacter-
iophages for fighting different bacterial infec-
tions around the world that the discipline
known as phage therapywas introduced.
Phage therapy rapidly developed globally and
was used as the major antibacterial in many
countries. Moreover, phage therapy also
attracted the attention of many pharmaceutical
companies including E.R. Squibb & Sons
(Princeton, NJ, USA), Eli Lilly (Indianapolis,
IN, USA) and Swan-Myers/Abbott laborato-
ries, which produced commercial phage prepa-
rations [4]. However, because phage properties
were poorly understood and well purified and
characterized preparations were lacking, phage
therapy resulted in variable outcomes and
many specialists questioned its efficacy. With
the introduction of antibiotics in the 1940s,
bacteriophages were unable to stand against the
miracle antibiotics [4]. However, during the
past two decades, which witnessed the continu-
ous rise in bacterial resistance together with
alarming decrease in the production of new
antibiotics, the Western world is actively revis-
iting the world of bacteriophages in different
practical applications. An interest that has
resulted in several companies developing
phage-based products for food, agriculture,
diagnostics and therapeutics (TABLE 1).
Bacteriophages: brilliant antibacterials
Bacteriophages are the most abundant micro-
organisms with an estimated number of
10
30
–10
32
phage particles. Humans are regu-
larly exposed to bacteriophages in water and
unprocessed food. Moreover, phages are pres-
ent in the intestinal tract, saliva and in dental
plaque. One milliliter of unpolluted water
contains 2 10
8
phage particles [5,6].
Bacteriophages infect bacteria via two possi-
ble lifecycles: the lysogenic and the lytic. The
infection begins with the adsorption of the
bacteriophage onto the surface of the bacteria
then the viral genetic material is injected into
the cytoplasm. Lytic bacteriophages take
immediate control of the biochemical machin-
ery of the host cell to make new virions and
the growth cycle ends by killing the host
cell [7]. In contrast, temperate bacteriophages
insert their genome into the chromosome of
the host cell and remain in the dormant state
until the bacteria is exposed to certain stimuli,
which lead to initiation of the lytic cycle.
TABLE 2illustrates attributes of bacteriophages
that make them attractive antibacterial thera-
peutics [8,9]. It is relatively easy and inexpensive
to find new phages against bacterial resistance
compared with years and billions of dollars in
case of antibiotic. Moreover, the ability of bac-
teriophages to kill pathogenic bacterial strains
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in situ through exponential growth of phage is significant for
the treatment of chronic bacterial infections [8,10].
Rediscovering bacteriophages
Since the discovery of penicillin, antibiotic production is esti-
mated at US$25 billion per year. The widespread and some-
times inappropriate use of antibiotics has contributed to the
increased incidence of bacterial resistance. The overuse of anti-
biotics in animal feed has also contributed to antibiotic resis-
tance. Many of the 17 antibiotic classes are used in animal
feed. Although Europe has banned the use of antibiotics for
promoting animal growth, it is still allowed in the USA [5].
In the USA, bacterial resistance costs the health-care system
over US$20 billion every year, leading to over 8 million extra
hospital days. Moreover, the social costs are over US$35 billion
every year. In the EU, multidrug-resistant bacteria result in the
death of nearly 25,000 patients annually and the associated
economical burdens are postulated to be nearly 1.5 billion
Euros annually [11].
Several antibiotic-resistant bacteria have been identified
including methicillin-resistant Staphylococcus aureus,
vancomycin-resistant S. aureus [8] and vancomycin-resistant
Enterococcus faecium [11]. In 2010, a serious Escherichia coli-
resistant strain was reported in India which carried a gene
called NDM1 (New Delhi metallo-b-lactamase). This was the
first report on spread of resistant strains in a community, as
such incidences were restricted to hospitals [12]. The problem of
bacterial resistance is further complicated with the marked
decrease in introducing new antimicrobials. Major decrease in
antibiotic development occurred over the past 25 years. From
2008 to 2012, only two antibiotics were under develop-
ment [13,14], and from 2003 to 2007, only five antibiotics were
underdevelopment compared with over 14 antibiotics back
between 1983 and 1987 [14].
Applications of bacteriophages
Although the focus of Western countries on bacteriophages as
an antibacterial has almost faded with the introduction of anti-
biotics, countries as the former Soviet Union with Georgia as
the epicenter continued their studies on bacteriophages to treat
serious and chronic infections [14].
In the 1980s, Smith and coworkers carried out several studies
to assess the effectiveness of phage therapeutics against E. coli
infections [1517]. Many other reports evaluated bacteriophage
therapeutic efficacy against different bacterial infections as E. coli
[18],Pseudomonas aeruginosa [19],Acinetobacter baumanii [20],
Klebsiella pneumoniae [21],Vibrio vulnificus [22], as well as Salmo-
nella [23] in animal models. Other studies revealed phage thera-
peutic effectiveness against drug-resistant strains of bacteria, such
as vancomycin-resistant E. faecium [24] and methicillin-resistant
S. aureus [25]. More remarkably, phages were able to reach intra-
cellular bacteria, such as Mycobacterium tuberculosis [26].
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
of biofilm formation [27,28]. Biofilms represent aggregation of
cells either eukaryotic or prokaryotic. Such cells are sur-
rounded by a matrix of extracellular polymeric substance. Bac-
teria found within biofilms possess increased resistance to
antibiotics and biocides, where it may require 1000-times
more of such agents to eradicate biofilm bacteria compared
with free-living bacteria [2 8]. Bacteriophages, however, have
proven ability to infect bacteria within biofilms [28]. Phage
replication within the bacterial cells results in localized ampli-
fication in phage numbers. Phages then spread within the bio-
film eradicating the bacteria producing the extracellular
polymeric substance. Moreover, some bacteriophages have the
ability to express or carry depolymerizing enzymes that can
degrade extracellular polymeric substance [28]. Unfortunately,
the chance of isolating a natural phage that shows both advan-
tages is low. Thus, researchers are now engineering phages to
express biofilm degrading enzymes [29]. Phage dispersing bio-
films show strong potential not only on the therapeutic front
but also at the industrial scale, where biofilms represent a con-
tinuous source of bacteria on different surfaces. It was shown
also that phages and antibiotics are not mutually exclusive
instead they could be synergistic, especially for managing bio-
film infections [30,31].
Table 1. List of some bacteriophage-based
companies.
Company Established Ref.
Omnilytics (USA) 1954 [98]
Exponential Biotherapies (USA) 1994 [99]
Biophage Pharma (Canada) 1995 [100]
PhageTech (Canada) 1997 [101]
Intralytix (USA) 1998 [102]
Hexal Gentech (Germany) 1998 [103]
Phage Biotech (Israel) 2000 [104]
GangaGen
(Bangalore, India; San Francisco,
CA; Ottawa, Canada)
2000 [105]
PhageGen
(Las Vegas, NV; previously Regma
Bio Technologies of London)
2000 [106]
Phico Therapeutics (UK) 2000 [107]
Phage-Therapy (Tbilisi, Georgia) 2002 [108]
Ampliphi biosciences (Australia) 2002 [109]
Novolytics (UK) 2002 [110]
Technophage (Portugal) 2002 [111]
Enzobiotics/New Horizons
Diagnostics (USA)
2003 [112]
Pherecydespharma (France) 2006 [113]
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Many studies were done on the use of phage as a microbial
control in food, including meat, poultry, fish, fruits and vegeta-
bles, both in the preharvest and postharvest (fresh and pack-
aged foods) stages. Studies have been conducted on different
stages in the food production chain, including also livestock
decontamination, and sanitation of contact surfaces and equip-
ment to control some of the most persistent foodborne patho-
gens in poultry including Campylobacter jejuni [32] and
Salmonella typhimurium [33]. Phage was also applied in cattle
and sheep against E. coli O157:H7 [34]. However, more studies
are required to enhance the in vivo biotherapeutic effects of
phages, including better understanding of the interactions
between phage and bacteria in the gut and alimentary system
of live animals [35].
Since the regulatory approval of ListShield
TM
as the first
phage-based product for control of Listeria in products of meat
and poultry, studies for phages targeting food (meat, fruits,
vegetables, processed ready to eat [RTE] food, cheese, pasteur-
ized milk, powdered infant formulas, etc.) in the postharvest
stage have increased [36]. Guenther et al. (2009 and 2012) stud-
ied the effect of bacteriophages against Listeria monocytogenes
and S. typhimurium in RTE foods, respectively [37,38].
Kim et al. (2007) reported the use of a phage cocktail against
Cronobacter sakazakii in reconstituted infant formula [39].
Anany et al. (2011) immobilized anionic heads of phages on
cationic cellulose membranes leaving the tails to catch and kill
both L. monocytogenes and E. coli O157:H7 in RTE and raw
meat [40].
Bacteriophages were also used as biocontrol agents in vegeta-
bles and fruits. Leverentz et al. (2001) used phage alone or in
combination with bacteriocin to reduce Salmonella contamina-
tion in fresh cut fruits [41]. Whereas, Viazis et al. (2010)
showed complete inactivation of E. coli O157:H7 strains in let-
tuce and spinach using a combination of phage mixture and
trans-cinnamaldehyde oil [42]. In 2013, the combined use of
bacteriophages with modified atmosphere packaging improved
the effect of bacteriophages [43].
Several reviews have addressed successful bacteriophage appli-
cations in human therapy [4446] and in food either in the pre-
harvest or postharvest stages [47].
Phages approved as biocontrol agents in the food
industry
The market of food and beverage was valued at US$5.7 trillion
worldwide in 2008 and is expected to reach US$7 trillion in
2014 [5]. Food poisoning and spoilage due to bacterial contam-
ination represent a major burden on health and food industry.
The Centers for Disease Control estimated about 9.4 million
cases of foodborne illnesses in addition to about 56,000 hospi-
talizations, and over 1350 deaths in the USA annually [48].
Moreover, microbial contamination is the major cause of food
spoilage, which results in loss of 25% of food produced each
year [49].
Due to the increase in bacterial resistance, together with the
progress achieved in understanding phage biology and its appli-
cations, numerous companies worldwide are currently investing
in developing phage-based products as food biocontrol agents,
decontamination, sanitation and diagnostics.
In 2005, the US Environmental Protection Agency approved
Agriphage
TM
(OmniLytics Inc., Sandy, UT) [50] for treating
bacterial spots in different crops. In 2006, the FDA approved
ListShield
TM
to be applied directly on RTE meat and poultry
to control L. monocytogenes [51]. Thus, bacteriophages were for
the first time considered generally recognized as safe
(GRAS) [29].TABLE 3presents selected examples of using
approved phage products in food.
In 2007, FDA approved phage-based preparations produced
by Omnilytics to decontaminate live animals from E. coli and
Salmonella [52]. Now, several products are commercially avail-
able including Finalyse spray against E. coli O157:H7 in cattle
and Armament against Salmonella in poultry [47].
The future of phage-based products in the food industry
shows tremendous promise, especially with the number of com-
panies as well as research institutes actively engaged in phage
Table 2. Comparison between phages and antibiotics as antibacterials.
Bacteriophages Antibiotics
Very specific affecting only the targeted bacterial species with no
disruption of normal flora therefore minimizing the possibility of
secondary infections
Target both pathogenic microorganisms and normal microflora
which may lead to serious secondary infections
Autodosing through replication at the site of infection (repeated
administration may not be needed)
Metabolized and eliminated and do not accumulate at the site
of infection
No serious side effects have been described
Minor side effects may be due to the liberation of endotoxins
from bacteria lysed in vivo by the phages
Multiple side effects, including allergies, intestinal disorders,
secondary infections, adverse effects on the kidney and the liver
Phage-resistant bacteria are not resistant to other phages having
a similar target range
Resistance to antibiotics extends over targeted bacteria
Finding new phages against developed bacterial resistance can
be achieved in days
Developing a new antibiotic against antibiotic-
resistant bacteria is a very lengthy and expensive process
Ability to clear biofilms Limited ability of biofilm clearance
Challenges facing phage therapy Review
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research. However, this is not the case for phage therapy in
humans, where a number of challenges still impede its
approval.
Phages not approved as therapeutics; challenges still in
the way
Despite the long history of phage therapy in addition to its
safety record, phages are still not approved by the FDA to be
used as therapeutics. Back in the 1970s, 1980s and 1990s, the
FDA revised the usage of phage preparations as therapeutics in
human. At the time, phage phiX174 was applied via intrave-
nous administration to patients with immunodeficiency such as
those infected with HIV. In the 1970s, after the inclusion of
phages in several vaccines, the FDA performed a safety review
on phages and concluded that bacteriophages are safe and
allowed the continued usage of the vaccines [7].
In 2008, the first phage Phase I clinical trial was approved
by the FDA. The study evaluated the use of a phage prepara-
tion of eight phages against venous leg ulcers. The trial verified
the safety of the phage cocktail preparation [53].
Despite the documented safety of bacteriophages, however,
regulatory bodies have not approved its use as a therapeutic,
although recently approving it in food. Such approval confirms
that phage is safe for human consumption and thus represents
a major step towards its approval as a therapeutic. Challenges
that currently hinder regulatory approval of phage as human
therapeutics as well as possible approaches to overcome them
will be discussed (FIGURE 1).
Immunogenicity
Phage immunogenicity is a major challenge facing phage ther-
apy, mainly because of its effects on phage pharmacokinetics
and also because of the possible side effects, such as
anaphylactic shocks. Phages are recognized as foreign antigens
by mammalian hosts [54].
It was initially reported that the reticulo-endothelial system
was responsible for the decrease in the concentrations of bacter-
iophages in the blood of mice [55]. Decades later it was proven
that the liver played the major role in phage elimination with
over 99% of the phages present in the circulatory system were
phagocytosed by the liver [56]. Such elimination of phage by
the reticulo-endothelial system, coinciding with lack of infor-
mation on phage pharmacokinetics, has resulted in majority of
phage applications being administered orally [57]. Consequently,
despite the advantageous therapeutic effects of phages on sys-
temic diseases, most studies to date were concerned with the
treatment of non-systemic diseases, such as gastrointestinal, ear
and wound infections [58]. Merril et al. [59] serially passed
phages in animals to isolate mutants that were capable of stay-
ing longer in circulation and allowing enhanced systemic
administration [59].
In addition to the interaction with the innate immune sys-
tem, bacteriophages also stimulate the adaptive immune system
and consequently the production of antibodies [60]. Less anti-
genic bacteriophages may be developed through phage display-
ing certain peptide ligands; Sokoloff and coworkers injected a
T7-phage peptide display library in rats, and observed that
phage displaying peptides with arginine or carboxy-terminal
lysine residues protected against the complement-mediated
inactivation of the bacteriophage by binding C-reactive protein,
whereas human serum phages that displayed C-terminal tyro-
sine residues were resistant to inactivation [61].
Within this context, Kim et al. were the first to introduce
PEGylation to bacteriophages to increase bacteriophages sur-
vival and efficacy [62]. Consistent with this, Molenaar et al.
have shown that the incorporation of targeting ligands
Table 3. FDA approved phage-based products for use in ready to eat food.
Product Regulatory approval Applications Ref.
ListShieldIntralytix, Inc.
USA
US FDA (2006) and USDA for direct application
onto foods (21 CFR 172.785.) EPA (EPA
registration 74234-1)
– Ready to eat food: salami, sausage,
basterami, etc.
– Sea food
– Food contact surfaces and enviroments
[114]
EcoShield Intralytix, Inc.
USA
FDA (2011) cleared as Food Contact Notification
or FCN, (FCN No. 1018). FSIS Directive
7120.1 (safe and suitable antimicrobial)
– Red meat parts and trim intended to be ground [115]
SalmoFresh Intralytix, Inc.
USA
FDA (GRAS Notice No. GRN 000435),
FSIS Directive the Star K-certified Kosher and
IFANCA-certified Halal product.
OMRI-listed suitable in the production of organic
foods
– Poultry
– Fish and shellfish
– Fresh and processed fruits and vegetables
[116]
LISTEXMicreos EBI
Food Safety
Netherlands
In 2006 approved by the FDA as GRAS, and by
the USDA in 2007 and by the EFSA, Health
Canada, BAG (Switzerland) and FSANZ (Food
Standards Australia New Zealand)
– Meat
– Ready to eat meat
– Fish
– Cheese
[117]
Agriphage Omnilytics
USA
EPA 2005 for use in agriculture – In agriculture on fruits and vegetables [118]
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increased the efficient delivery and specificity of the
M13 phage to liver Kupffer and parenchymal cells [63].
S-radiolabeled M13 bacteriophage was chemically modified by
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
purpose of this study was to increase efficiency of in vivo pan-
ning of phage libraries, the same principle could be used for
targeting other organs for therapeutic purposes [63].
Pharmacokinetics
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
effects. Phages replicate exponentially, interact and show differ-
ent phenomena completely different from the chemical kinetics
of conventional drugs [64]. Consequently, studying the pharma-
cokinetics of bacteriophages as antibacterial therapeutics necessi-
tates knowledge of the infected host, the bacteriophage and the
infecting bacteria as well as their complex interactions. Interest-
ingly, through the process of infection and phage therapy, the
growth of bacteria would lead to exponential growth of phages,
unlike the conventional use of antibiotics [64]. Along this line,
Dubos and coworkers treated mice that were infected intracere-
brally with Shigella dysenteriae by administering phage into the
peritoneal cavity [65]. Similar studies by Smith and Huggins
demonstrated that mice infected either intracerebrally or intra-
muscularly with E. coli were rescued with phages via intramus-
cular administration [15]. In both the studies, the levels of the
bacteriophages were highest in the infected tissues but then
decreased as the bacterial levels decreased. On the other hand,
phage levels in the blood of the control animals were in accor-
dance with the phage dilution in the total blood of the animal.
These data were used as the basis for mathematical models pre-
dicting the kinetic behavior of phages [64,66]. Several parameters
should be considered to achieve effective phage therapeutics.
These include the route and timing of administration and the
required dose [64].
Systemic side effects
Lytic phages are the only phages that are used in therapy. First
because they are more potent than the temperate phages, and
second because temperate phages have the risk of transferring
fragments of the DNA of the host to other bacterial species,
thus posing the risk of producing virulent strains if such frag-
ments were toxin encoding or antibiotic-resistant genes.
No reports have been recorded of serious complications
accompanying phage therapy. Moreover, humans are in contin-
uous exposure to bacteriophages, as they are so common in the
environment. For example, unpolluted water contains ca. 2
10
8
(phage/ml), and phages are continuously consumed in
foods [44].
Safety of bacteriophages was proved through different studies
where bacteriophage did not cause adverse effects in
humans [67,68]. However, there are concerns that any lytic
bacterial treatment will generate endotoxins and superantigens
that may be released during the massive and fast destruction of
bacteria in vivo [58]. Although this possibility may be low, engi-
neered phages that are either lysis deficient or non-replicative
have been developed. For example, filamentous phages were
engineered to express certain toxic genes in E. coli leading to
bacterial toxicity but not their lysis, thus overcoming the prob-
lem of endotoxins [69]. Non-lytic and non-replicative phages
were also designed against P. aeruginosa [70]. Interestingly, in
both the studies, it was observed that the non-lytic phages
increased the survival of treated infected mice compared with
lytic phages, which may have been caused by reduced
inflammation.
It is also important to ensure that bacteriophages for ther-
apy do not carry gene sequences that possess significant simi-
larity with antibiotic-resistance genes, genes for other bacterial
virulence factors and genes for phage-encoded toxins. More-
over, phages should be also incapable of generalized transduc-
tion [47]. A number of quality assurance and quality control
measures to consider are described in Merabishvili et al.
(2009) [71].
Narrow host range
Each phage strain infects only one type of bacteria. The down-
side of such narrow host range is the limited applicability in
cases where the identification of the specific bacterial host can-
not be achieved [7]. However, with the tremendous develop-
ments of rapid diagnostics, identifying the specific bacterial
host is easily achieved. In most situations, however, bacterio-
phage cocktails are used to broaden the host range and increase
the overall therapeutic efficiency. It is unlikely, however, to
achieve 100% therapeutic efficiency [7,58]. Thus, the use of sin-
gle phages versus cocktails of phages has to be further studied
and optimized as both regimens may lead to the development
of resistance [72]. Nevertheless, as will be discussed later,
approval of phage cocktails is problematic, thus other methods
have also to be developed. Using broad host spectrum bacterio-
phages, such as Listeria P100 or S. aureus phi812 was pro-
posed [73,74]. Also, broad host range could be achieved via
Regulatory
approval
Bacterial
resistance
Immunogenicity
Manufacturing
Challenges
facing phage
therapy
Systemic side
effects Patentability
Narrow host
range
Pharmacokinetics
Figure 1. Challenges facing phage therapy.
Challenges facing phage therapy Review
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grafting the g3p phage protein of one filamentous bacterio-
phage to another [75,76]. Such an approach, however, although
would broaden the host range of the engineered bacteriophage
and would facilitate its patentability, may also complicate its
regulatory approval. Moreover, safety of such engineered
phages has not been thoroughly studied, especially that such
engineered phages may increase the probability of phage-
mediated transfer of genetic sequences among bacterial
species [77].
Developing clinical diagnostics to allow rapid identification
of the infective bacterial pathogen as well as their phage suscep-
tibility may be necessary to allow the use of the required phage
instead of phage cocktails. These phage-based diagnostics
should be fast and reliable [78].
Along this line, Loessner and coworkers [78] developed lucif-
erase reporter bacteriophages for the sensitive and fast detection
of viable cells of Listeria. In addition, diagnostic tests that uti-
lized phage enzyme lysins have been developed allowing rapid
bacterial identification and have resulted in commercialized
products (Enzobiotics; Columbia, MD, USA) [9].
Bacterial resistance
Bacteriophages are the most abundant and diverse microorgan-
isms on the planet. Such diversity is due to the continuous
adaptation to different pressures. Bacteriophages have evolved
different mechanisms that enable it to avoid, withstand and get
over different mechanisms of bacterial resistance [79]. It was
found that in monoculture studies performed in vitro, phage
resistance can evolve within hours or days [58]. However, it was
observed that bacteria develop phage resistance in vitro more
rapidly than in vivo [80]. Thus, the correlation between the rate
of resistance development in vivo and in vitro is an area that
requires further study.
The exposure of a certain bacterial strain to a single bacterio-
phage is suggested to aid in the emergence of phage-resistant
strains of the bacteria. On the other hand, several studies report
that phage cocktails help control or delay the evolution of
phage-resistant strains [72,81,82]. Reduction of appearance of
resistant bacterial strains can be achieved using new techniques,
such as using combinations of bacteriophages with other anti-
microbials (such as antibiotics or antimicrobial herbal extracts),
cycling bacteria through mixtures of different phages and tar-
geting phage-resistance mechanism by genetically engineered
bacteriophages [58].
In this context, additional studies are needed to identify
phage receptors and understanding the interaction of bacterio-
phages with the receptors on the host surface. Until now,
except for E. coli, very few phage receptors have been identi-
fied [79]. Moreover, the evolution of phages in response to the
mutations in the host surface receptors needs to be investi-
gated. Finally, the environment in which phage-resistance
mechanisms are studied need to be translated from a closed
laboratory environment with single phage at a time to a more
real complex environment containing multiple anti-phage
barriers [79].
Manufacturing; (safety & efficacy)
In the past, and in Russia until now, phages have been admin-
istered to humans: orally, either in tablets or liquid formula-
tions (10
5
to 10
11
PFU/dose); locally to the skin, ear or eye in
the form of creams, tampons and rinses; rectally; as aerosols; as
intrapleural injections; and intravenously [45]. Still, there are
two critical factors that need to be standardized during bacteri-
ophage manufacturing, which are safety and efficacy. Inability
to optimize these factors may have been responsible for the
recorded negative results of phage therapy. Efficacy of the
phage preparation reflects the presence of sufficient virulent
phages, especially against the target bacteria [83]. Poor viability
and/or stability of phage preparations may result from
improper manufacturing techniques. Therefore, in some early
commercial phage preparations, mercurials and oxidizing agents
as well as heat treatments were used to ensure bacterial sterility.
Nevertheless, such treatments may result in ineffective prepara-
tions due to phage inactivation. However, advanced
manufacturing techniques are now utilized for phage purifica-
tion. Moreover, determination of the phage titer and viability
is essential [83].
Safety on the other hand is a very important parameter, as
issues related to the presence of exotoxins and pyrogens released
during bacterial lysis by bacteriophages have always raised seri-
ous safety concerns. Consequently, bacteriophage manufacturing
for clinical use must be optimized to eliminate bacterial exotox-
ins and pyrogens [60]. Early therapeutic phage preparations suf-
fered from inefficient purification resulting in crude lysates of
the host bacteria in the preparation [84]. Efficient purification
can be achieved by ammonium sulfate precipitation and also by
CsCl gradient centrifugation [84]. Ultrafiltration and two-step
chromatography were also used [84]. However, the purification
procedures have witnessed considerable evolution in the past few
years. Recently, Kramberger and coworkers [85] utilized methac-
rylate monoliths columns in S. aureus phage purification from a
lysate of bacterial cells. This method resulted in recovery of
60% of viable phages in a single purification step [85]. In this
context, Gill and Haymen, published an interesting review
exploring the different available methods for bacteriophage puri-
fication as well as the different levels of purification required for
different applications together with the current protocols utilized
in the commercial production of different products of phage
therapy [83]. Standard purification techniques should be devel-
oped to achieve clinical grade bacteriophage preparations.
Regulatory approval
Despite the successful application of phage therapy for decades
in some eastern countries such as Poland and the former Soviet
republics, especially Georgia and Russia [86], their clinical data
failed to gain regulatory approval because such data were not
developed under the regulatory authorities frameworks.
A major hurdle in the way of phage becoming part of the
mainstream medicine is the absence of well-defined guidelines
for regulatory approval of phage. Instead, phage is regulated
according to existing guidelines developed for typical
Review Sarhan & Azzazy
96 Expert Rev. Anti Infect. Ther. 13(1), (2015)
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For personal use only.
antibacterials [7], meaning that every component of the thera-
peutic cocktail of phages must undergo individual clinical trials
and that the composition of the approved phage cocktail can-
not be changed without re-approval. Such regulations do not
take into consideration the differences between phages and
antibiotics. Fortunately, there exists a regulatory framework
that could be applied for phage therapy, which is that of the
influenza live-virus vaccine. Such a vaccine is formulated from
a cocktail of three or four influenza strains that the FDA has
approved to be reformulated annually according to the circulat-
ing flu strain [7].
However, with the recent approval of different phage prepara-
tions in food and agriculture together with the approval of some
clinical trials, the situation may be improving. Recently some
Phase I studies have been carried out and published [87], whereas
others are being conducted but to date have not been pub-
lished [53]. In 2009, Biocontrol Ltd Company (Nottingham,
UK) has completed randomized, double-blind, placebo-con-
trolled, fully regulated Phase II clinical trial for phage therapy
against Pseudomonas infections in the western world [54]. Another
FDA-approved Phase I clinical trial in 2008 used phage to target
P. aeruginosa, E. coli, and S. aureus in venous ulcers [87].
An important approach that should be considered by the
regulatory authorities is regulating phages for the pharmacy
approach. The approach allows phage cocktails to be custom
made by licensed hospital pharmacists from current Good
Manufacturing Practice phage preparations instead of all hospi-
tals using the same preparations [7].
In Europe, and pending resolution of regulatory issues, phage
therapy is used under the supervision and responsibility of medi-
cal ethical committees within the approval of the World Medical
Association Declaration of Helsinki [88,89]. This is only a tempo-
rary solution that will not result in efficient introduction of
phage therapy into the western or the rest of the world [53].
Patentability
Recently, a patent by Heo and coworkers was granted and was
based on bacteriophage MPK6, a novel bacteriophage belong-
ing to the order Caudovirales, and its pharmaceutical composi-
tion for treating P. aeruginosa [90,91]. The issue of phage
patentability resembles that of patenting monoclonal antibod-
ies, where in both cases a wide array of potential targets exist
and the methodology for their discovery is common knowl-
edge. Individual bacteriophages are patentable where they can
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
and optimized to formulate even more efficient bacteriophage
preparations [53]. The European patent laws allow patenting of
known phages provided that the use of these phages has not
been disclosed [93]. Moreover, the new phage formula can be
patented [53]. The European patent office necessitates a certain
technical intervention for isolating phage from its natural envi-
ronment, as well as proper characterization of the isolated
phage. For the US Patent and Trademark Office, however,
only genetically engineered bacteriophages can meet the patent
regulations. Several patents, however, have been granted by the
US patent office, including a patent for reduction of sepsis
with a pharmaceutical preparation including bacteriophages, as
well as other patents approved for using phage in the food sec-
tor. A detailed discussion of patent issues related to therapeutic
use of phage is described elsewhere [9395].
Expert review
The regulatory approval of phage consumption in food pro-
vides a validation for its safety for humans. Thus, the main
hurdles still facing the regulatory approval of phage as thera-
peutics include the need for more clinical evidence for its thera-
peutic efficacy as well as new or modified regulatory guidelines.
Such clinical evidence must be supported by detailed data on
phage immunogenicity, pharmacokinetics and formulations.
Some approaches for overcoming the challenges that hinder
the approval of such an important therapeutic are discussed
below.
.Bacteriophages act as exogenous antigens inducing a humoral
reaction due to the presence of immunogenic epitopes in
phage proteins [64]. Masking or modifying these recognition
epitopes is needed to improve phage blood circulation and
decrease immunogenic response. Chemical modification
through PEGylation of the bacteriophages and/or addition of
biocompatible moieties can be used to improve phage phar-
macokinetics and decrease its immunogenicity [61,63].
.Phage pharmacokinetics. The basic kinetic principles of self-
replicating phages have been recently addressed in a number
of important studies [7,64] which will allow better experimen-
tal design and data interpretation. There is still a need to
develop kinetic models describing the density dependent bac-
teria–phage interactions [8].
.Optimization of the formulation and long-term stability. A
small-scale quality controlled production of defined bacterio-
phage cocktail for use in human was developed and resulted
in approved clinical trials [71].
.The approval of phage in food (TABLE 1) should motivate
researchers and the pharmaceutical industry to pursue more
clinical evidence for its therapeutic efficacy and to work with
the regulatory agencies to develop new guidelines for phage
therapy. This is now important with the alarming rise of bac-
terial resistance and the low number of new antibiotics
introduced [86].
Five-year view
The application of bacteriophages in different food sectors is
expected to increase supported by the regulatory approval of
different phage products (TABLE 1).
With the alarming increase in bacterial resistance together
with the decreased number of new antibiotics, the current regu-
latory guidelines may be modified in favor of phage therapy.
Developing specific and simple tests that allow rapid identifi-
cation of the causative bacterial strain as well as their phage
Challenges facing phage therapy Review
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susceptibility is one of the major areas that will aid in phage
adoption as therapeutics. Along this line, nano-based tests will
be one of the fast developing areas in the upcoming years [96].
It is expected that stable phage formulations alone or with
other antibiotics or natural antibacterials may be developed
with the aid of novel nanomaterials [97].
Financial & competing interests disclosure
W Sarhan and H Azzazy are authors of a patent application describing
the use of nanofibers and phage for wound healing. The authors have no
other relevant affiliations or financial involvement with any organization
or entity with a financial interest in or financial conflict with the subject
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.
.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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Papers of special note have been highlighted as:
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Challenges facing phage therapy Review
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  • ... The Earth's biosphere is estimated to contain approximately 10 32 phage particles, exceeding the number of prokaryotic organisms by 10-fold [33,41,42]. Bacteriophages differ in size and shape, and are characterized with incredible genome mosaicity resulting from different environmental pressures, including the adaptation to continuous bacterial resistance [43]. At the time of writing this article, only 8852 complete genomic sequences have been listed in the NCBI database of phage genomes. ...
    Article
    Full-text available
    Bacteriophages (phages) are the most abundant and diverse biological entities in the biosphere. Due to the rise of multi-drug resistant bacterial strains during the past decade, phages are currently experiencing a renewed interest. Bacteriophages and their derivatives are being actively researched for their potential in the medical and biotechnology fields. Phage applications targeting pathogenic food-borne bacteria are currently being utilized for decontamination and therapy of live farm animals and as a biocontrol measure at the post-harvest level. For this indication, the United States Food and Drug Administration (FDA) has approved several phage products targeting Listeria sp., Salmonella sp. and Escherichia coli. Phage-based applications against Campylobacter jejuni could potentially be used in ways similar to those against Salmonella sp. and Listeria sp.; however, only very few Campylobacter phage products have been approved anywhere to date. The research on Campylobacter phages conducted thus far indicates that highly diverse subpopulations of C. jejuni as well as phage isolation and enrichment procedures influence the specificity and efficacy of Campylobacter phages. This review paper emphasizes conclusions from previous findings instrumental in facilitating isolation of Campylobacter phages and improving specificity and efficacy of the isolates.
  • ... To obtain regulatory approvals, current research is heavily exploring roadblocks through numerous animal studies and clinical trials [82][83][84]. The FDA has, however, approved the use of phages for food decontamination [85], dietary supplements [86], and environmental prophylaxis. Phage is allowed only on a compassionate care basis for human therapeutic use [87,88]. ...
    Article
    Full-text available
    Bacteriophages are viruses that infect bacteria. After their discovery in the early 1900s, bacteriophages were a primary cure against infectious disease for almost 25 years, before being completely overshadowed by antibiotics. With the rise of antibiotic resistance, bacteriophages are being explored again for their antibacterial activity. One of the critical apprehensions regarding bacteriophage therapy, however, is the possibility of genome evolution, development of phage resistance, and subsequent perturbations to our microbiota. Through this review, we set out to explore the principles supporting the use of bacteriophages as a therapeutic agent, discuss the human gut microbiome in relation to the utilization of phage therapy, and the co-evolutionary arms race between host bacteria and phage in the context of the human microbiota.
  • Article
    Background: The gut microbiota plays an important role in the pathogenesis of several gastrointestinal diseases. Its composition and function are shaped by host-microbiota and intra-microbiota interactions. Bacteriophages (phages) are viruses that target bacteria and have the potential to modulate bacterial communities. Aims: To summarise phage biology and the clinical applications of phages in gastroenterology METHODS: PubMed was searched to identify relevant studies. Results: Phages induce bacterial cell lysis, integration of viral DNA into the bacteria and/or coexistence in a stable equilibrium. Bacteria and phages have co-evolved and their dynamic interactions are yet to be fully understood. The increasing need to modulate microbial communities (e.g., gut microbiota, multidrug-resistant bacteria) has been a strong stimulus for research in phages as an antibacterial therapy. In gastroenterology, phage therapy has been mainly studied in infectious diseases such as cholera. However, it is currently being explored in several other circumstances such as treating Clostridioides difficile colitis, targeting adherent-invasive Escherichia coli in Crohn's disease or eradicating Fusobacterium nucleatum in colorectal cancer. Overall, phage therapy has a favourable and acceptable safety profile. Presently, trials with phage therapy are ongoing in Crohn's disease. Conclusions: Phage therapy is a promising therapeutic tool against pathogenic bacteria in the fields of infectious diseases and gastroenterology. Randomised, placebo-controlled trials with phage therapy for gastroenterological diseases are ongoing.
  • Article
    Full-text available
    Bacteriophages are viruses that are ubiquitous in nature and infect only bacterial cells. These organisms are characterized by high specificity, an important feature that enables their use in the food industry. Phages are applied in three sectors in the food industry: primary production, biosanitization, and biopreservation. In biosanitization, phages or the enzymes that they produce are mainly used to prevent the formation of biofilms on the surface of equipment used in the production facilities. In the case of biopreservation, phages are used to extend the shelf life of products by combating pathogenic bacteria that spoil the food. Although phages are beneficial in controlling the food quality, they also have negative effects. For instance, the natural ability of phages that are specific to lactic acid bacteria to destroy the starter cultures in dairy production incurs huge financial losses to the dairy industry. In this paper, we discuss how bacteriophages can be either an effective weapon in the fight against bacteria or a bane negatively affecting the quality of food products depending on the type of industry they are used.
  • Article
    Metal based nanocomposites are gaining popularity for the past few years due to their promising chemical and physical properties. These nanocomposites can be obtained by incorporation of metal nanoparticles with glass, ceramic and polymer. Metal polymer nanoparticles can be formed through direct reduction method, in situ methods like chemical reduction, photoreduction and thermal decomposition of metallic salt inside the polymer, ex-situ by direct insertion of metallic nanoparticles into the polymer, through vapor phase deposition techniques and ion implantation. Natural polymers such as cellulose, starch, chitin, chitosan, gelatin, dextran, alginate, pectin, guar gum, rubber and fibrin are preferred than the synthetic ones due to their amazing properties including maximized purity and crystallinity, tensile solidity, improved elasticity and extensive surface area. In our review, we spotlight the fabrication methods and the innovative applications of many natural polymers metal nanocomposites, as well as their antibacterial efficacy against Escherichia coli and Staphylococcus aureus.
  • Chapter
    The rise of antibiotic-resistant bacterial strains is a global concern in many sectors, such as aquaculture, as described in chapter “The Rise and Fall of Antibiotics in Aquaculture.” To counter this phenomenon, several alternatives or complement to antibiotics have been investigated. Here, we will look at one of those proposed strategies that of using bacteria-specific viruses, called bacteriophages, or commonly phages. Since their discovery in the early 1900s, bacteriophage treatments have had a fleeting popularity in Western countries due to several scientific reasons as well as in some cases, political motives. Only recently, with the appearance of multidrug-resistant bacterial strains, a new craze for phage therapy appeared in Western countries. In an aquaculture context, some studies have shown promising results for the treatment of fish diseases using phages. More specifically, the experimentations with phage cocktail against A. salmonicida, infectious agent of furunculosis in salmonids, both in vitro and in vivo, provide an interesting foundation for future alternative treatments. However, since phages and bacteria are evolving entities, this biological war is far from over. The presence of phage-resistance mechanisms in bacteria and other technical aspects of phage therapy in aquaculture are factors to consider before having any applicable treatments.
  • Article
    Full-text available
    Enterococcus faecium, is an important nosocomial pathogen with increased incidence of multidrug resistance (MDR) – specifically Vancomycin resistance. E. faecium constitutes the normal microbiota of the human intestine as well as exists in the hospitals and sewage, thus making the microorganism difficult to eliminate. Phage therapy has gained attention for controlling bacterial MDR infections and contaminations. We have successfully isolated from waste water and characterized a lytic bacteriophage STH1 capable of targeting Vancomycin resistant Enterococcus faecium (VREF) with high specificity. The phage was isolated from sewage water of a hospital at district Dera Ismail Khan, Pakistan. Initial characterization showed that magnesium and calcium ions significantly increased phage adsorption to the host. One step growth experiment showed a latent period of 18 min with burst size of 334 virions per cell. Optimal temperature and pH of the phage was 37°C and 7.0, respectively. Phage application to host strain grown in milk and water (treated and untreated) showed that the phage efficiently controlled bacterial growth. The study suggests that the phage STH1 can serve as potential control agent for E. faecium infections in medical facilities and in other environmental contaminations.
Literature Review
  • Article
    Full-text available
    The worldwide emergence of 'superbugs' and a dry antibiotic pipeline threaten modern society with a return to the preantibiotic era. Phages - the viruses of bacteria - could help fight antibiotic-resistant bacteria. Phage therapy was first attempted in 1919 by Felix d'Herelle and was commercially developed in the 1930s before being replaced by antibiotics in most of the western world. The current antibiotic crisis fueled a worldwide renaissance of phage therapy. The inherent potential of phages as natural biological bacterium controllers can only be put to use if the potential of the coevolutionary aspect of the couplet phage-bacterium is fully acknowledged and understood, including potential negative consequences. We must learn from past mistakes and set up credible studies to gather the urgently required data with regard to the efficacy of phage therapy and the evolutionary consequences of its (unlimited) use. Unfortunately, our current pharmaceutical economic model, implying costly and time-consuming medicinal product development and marketing, and requiring strong intellectual property protection, is not compatible with traditional sustainable phage therapy. A specific framework with realistic production and documentation requirements, which allows a timely (rapid) supply of safe, tailor-made, natural bacteriophages to patients, should be developed. Ultimately, economic models should be radically reshaped to cater for more sustainable approaches such as phage therapy. This is one of the biggest challenges faced by modern medicine and society as a whole.
  • Article
    The ability to preserve and deliver reagents remains an obstacle for the successful deployment of self-contained diagnostic microdevices. In this study we investigated the ability of bacteriophage T7 to be encapsulated and preserved in water soluble nanofibers. The bacteriophage T7 was added to mixtures of polyvinylpyrrolidone and water and electrospun onto a grounded plate. Trehalose and magnesium salts were added to the mixtures to determine their effect on the infectivity of the bacteriophage following electrospinning and during storage. The loss of T7 infectivity was determined immediately following electrospinning and during storage using agar overlay plating and plaque counting. The results indicate that the addition of magnesium salts protects the bacteriophage during the relatively violent and high voltage electrospinning process, but is not as effective as a protectant during storage of the dried T7. Conversely, the addition of trehalose into the electrospinning mix has little effect on the electrospinning, but a more significant role as a protectant during storage.
  • Article
    Full-text available
    Despite advances in modern technologies, the food industry is continuously challenged with the threat of microbial contamination. The overuse of antibiotics has further escalated this problem, resulting in the increasing emergence of antibiotic-resistant foodborne pathogens. Efforts to develop new methods for controlling microbial contamination in food and the food processing environment are extremely important. Accordingly, bacteriophages (phages) and their derivatives have emerged as novel, viable, and safe options for the prevention, treatment, and/or eradication of these contaminants in a range of foods and food processing environments. Whole phages, modified phages, and their derivatives are discussed in terms of current uses and future potential as antimicrobials in the traditional farm-to-fork context, encompassing areas such as primary production, postharvest processing, biosanitation, and biodetection. The review also presents some safety concerns to ensure safe and effective exploitation of bacteriophages in the future. Expected final online publication date for the Annual Review of Food Science and Technology Volume 5 is February 28, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
  • Article
    Full-text available
    Bacteriophage-based medical research provides the opportunity to develop targeted nanomedicines with heightened efficiency and safety profiles. Filamentous phages also can and have been formulated as targeted drug-delivery nanomedicines, and phage may also serve as promising alternatives/complements to antibiotics. Over the past decade the use of phage for both the prophylaxis and the treatment of bacterial infection, has gained special significance in view of a dramatic rise in the prevalence of antibiotic resistance bacterial strains. Two potential medical applications of phages are the treatment of bacterial infections and their use as immunizing agents in diagnosis and monitoring patients with immunodeficiencies. Recently, phages have been employed as gene-delivery vectors (phage nanomedicine), for nearly half a century as tools in genetic research, for about two decades as tools for the discovery of specific target-binding proteins and peptides, and for almost a decade as tools for vaccine development. As phage applications to human therapeutic development grow at an exponential rate, it will become essential to evaluate host immune responses to initial and repetitive challenges by therapeutic phage in order to develop phage therapies that offer suitable utility. This paper examines and discusses phage nanomedicine applications and the immunomodulatory effects of bacteriophage exposure and treatment modalities.
  • Article
    Phage therapy is the use of bacterial viruses (bacteriophage) to treat bacterial infections. It has been practiced sporadically on humans and domestic animals for nearly 75 yr. Nevertheless, phage therapy has remained outside the mainstream of modern medicine, presumably because of doubts about its efficacy, and possibly because it was eclipsed by antibiotics and other chemotherapeutic agents. In this report, we develop the study of phage therapy and antibiotic therapy as a population biological phenomenon-the dynamic interaction of bacteria with a predator (phage) or a toxic chemical (antibiotic) inside a host whose immune and other defenses also affect the interaction. Our goal is to identify the conditions under which phage and antibiotics can successfully control a bacterial infection and when they cannot. We review data published in the 1980s by H. Williams Smith and J. B. Huggins on the use of phage and antibiotics to control lethal, systemic infections of Escherichia coli in experimentally inoculated mice. We show that some of their observations can be accommodated by a quantitative model that invokes known or plausible assumptions about host defenses and the interactions of bacteria with phage and antibiotics; some observations remain unexplained by the model. Our analysis identifies several hypotheses about the population dynamics of phage and antibiotic therapy that can be tested experimentally. Included among these are hypotheses that account for variation in the efficacy of the different phages employed by Smith and Huggins and why, in their study, phages were more effective than antibiotics.