Bacteriophage therapy – cooked goose or Phoenix rising?
Michael Mattey1 and Janice Spencer2
1
University of Strathclyde
Strathclyde Institute of Pharmacy and Biomedical Sciences
Royal College Building
204, George Street
Glasgow G1 1XW
Scotland, UK
2
Blaze Venture Technologies Limited
Broadmeads, Ware,
Hertfordshire SG12 9HS,
UK.
Summary:
Recent animal and human trials of bacteriophage therapy have demonstrated its potential
to alleviate bacterial diseases, both in internal and external applications. The regulatory
requirements are becoming clearer as more examples are presented. A core of GLP
(Good Laboratory Practice) studies will be needed to validate safety and clinical trials to
validate efficacy. GMP (Good Manufacturing Practice) production requirements and
quality issues will mean comparable costs to the production of conventional antibiotics
should be anticipated. The definition of the “active substance” will be central to the
success of bacteriophages therapy to ensure the variety and evolutionary potential of
bacteriophages are exploited.
Bacteriophage therapy
Introduction:
Bacteria evolved long before humans appeared and we have co-existed with them
throughout our own evolution. The bacterial disease that infected our ancient ancestors,
tuberculosis, leprosy, plague, cholera, typhoid, and typhus still cause deaths today.
The “Black Death” of 1347 killed about 25 million people in Europe in 5 years, which
was between a quarter and half the population. Even now, with antibiotics widely
available, a proportion of the 3.9 million deaths from pneumonia each year are bacterial,
while tuberculosis kills about 1.6 million and diarrhoeal diseases account for another 1.6
million. Since the start of antibiotic treatment in the 1940’s bacterial infections resistant
to antibiotics have increased and over the past few years have swamped health services
with difficult to treat and manage infections. This has lead to increased hospital stays,
costs and mortality.
The Arabic physician Avicenna, in the early 11th century, is often credited with
discovering the contagious nature of diseases and he introduced the concept of
quarantine as a means of limiting the spread of disease. [1], a concept reflected in the
successful use of individual isolation rooms for patients with hospital acquired infections.
Treatments for bacterial and other diseases were historically based on plants, animal or
inorganic materials although fungal antibiotics were unknowingly employed. Equally,
bacteriophages were probably utilised; the practice of drinking natural “holy waters” to
cure “consumption” and other diseases, the concept of baptism by immersion, originally
in rivers (Jordon and Ganges!), leading to “miracle cures” may have a rational basis in
bacteriophages. Mud (a source of adsorbed bacteriophages) or clay poultices have long
been used for skin complaints, the earliest record dates from the last quarter of the third
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Bacteriophage therapy
millennium BC; prescription 5 of a medical cuneiform tablet reads “Pulverise river mud
and..…knead it with water; rub with crude oil and fasten as a poultice.” [2].
Microorganisms have contested resources for billions of years, bacteria appeared about
4000 Myr ago and their viruses, bacteriophages, may have evolved soon after as part of
the bacterial struggle for resources, with a virus that infected a competitor being
advantageous. Antibiotics were a later fungal or bacterial weapon, their producers
appearing as recently as 1000Myr. When we ask the question “antibiotic or bacteriophage
therapy?” we seek to utilise the weapons of a very ancient war.
History of bacteriophages:
Bacteriophages were officially discovered by Felix d’Herelle in 1917, a French-Canadian
microbiologist at the Institut Pasteur in Paris, but the existence of bacteriophage was
hypothesised by Frederick Twort a few years earlier [3]. The subsequent history of
success, failure and politics has been extensively reviewed [4 -18].
Microbiology:
Bacteriophages constitute a group of viruses with the ability to invade various bacterial
species as their specific hosts. They are widespread in nature and are known to attack
over 140 bacterial or archaeal genera. Moreover, bacteriophages are universally observed
in open and coastal waters, marine sediments, terrestrial ecosystems such as soil, and the
bodies of humans, animals, and insects [19]. Their presence has been detected in faeces
[20], saliva [21, 22], human and bovine serum and they are known to be in high
abundance in aquatic environments [23]; non-polluted water contains 2 x 108
bacteriophages per ml. It is thought that for each individual bacterial cell there are ten
bacteriophage particles [12].
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Bacteriophage therapy
According to the International Committee on Taxonomy (ICTV), bacteriophage can be
divided into 13 families and 30 genera, each with a variety of distinguishing
characteristics, such as size, nature of genetic material and appearance. The “mosaic
concept” suggests that each bacteriophage is defined by its own set of properties based on
related genetic elements [19]. The majority of bacteriophages contain dsDNA, although a
minority contain ssDNA, ssRNA, or dsRNA.
Once a bacteriophage has infected the host cell, the resultant infection may be either lytic,
reprogramming and soon destroying the infected cell, or lysogenic, where the
bacteriophage genome is integrated into the bacterial genome and passed on to future
generations of bacteria.
Lytic bacteriophages are used for most therapeutic approaches. A typical lytic
bacteriophage will produce 100-300 new bacteriophages from an infected bacterial cell in
a matter of hours. These can go on to infect and kill a new generation of the target
bacteria. Exponential replication only stops when the bacteriophages run out of target
bacteria. Without their target, they are simply small lumps of protein, potential nutrients
for other microorganisms, which would be removed from humans by the normal
clearance processes of the body.
Bacteriophage Therapy, what is it?
Therapy is simply the medical treatment of disease so that bacteriophage therapy covers a
wide range from the treatment of external and internal bacterial infections with intact
natural bacteriophages or genetically modified bacteriophages [24] to the use of
bacteriophage components [25]. This article covers only the use of natural intact
bacteriophages.
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Bacteriophage therapy
Bacteriophage therapy vs. chemotherapy
Almost every review on bacteriophage therapy cites a number of “advantages” of
bacteriophages over antibiotics but most such reviews are written by proponents of
bacteriophage therapy. Over optimism that is not supported by clinical evidence and
undue pessimism or conspiracy theories are not helpful. The introduction of
bacteriophage therapy will require a combination of efficacy and commercial value and
the present situation in much of the world is that antibiotic treatment is the only therapy
available. To displace a dominant product in any market requires a medium to long term
strategy, a rapid displacement is unlikely. The experience of bacteriophage therapy in a
command economy such as that existing in Eastern Europe until recently is not a useful
model; regulatory requirements and cost effectiveness will not be waived because
bacteriophages are different from the products of the chemical industry.
Advantage one: “Bacteriophages only kill harmful pathogens”.
The specificity of bacteriophages varies from a few extremely selective bacteriophages,
often used in bacteriophage typing of bacteria, to relatively broad specificity
bacteriophages, but none have the breadth of action of antibiotics. This is indeed
advantageous in that unrelated bacteria are not killed by bacteriophages, so that for
example, gut bacterial flora are not unnecessarily disrupted by therapy, but is a lot less
relevant in septacaemia when the presence of any bacteria is undesirable. Where a
bacterial infection is acute a broad spectrum antibiotic must be the clinical choice for
rapid treatment but for chronic infection, where antibiotics have failed to eliminate the
infection and the patient is still alive, then bacteriophages may have a role.
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Bacteriophage therapy
Although much has been written, particularly in the media, about the end of the antibiotic
era, it is likely that antibiotics will continue to be the first line treatment for the near and
mid term future. It is worth noting that antibiotic resistance rarely exceeds 70% of the
clinical isolates which suggests that the evolutionary pressures towards specific antibiotic
resistance is countered by other factors. Since the mode of action of bacteriophages is
different to that of any antibiotic the ability of bacteriophages to relieve or counter the
evolutionary pressure of antibiotics on bacteria must count as an advantage.
Advantage two: “Bacteriophages are active against antibiotic resistant bacteria”
How much of a problem antibiotic resistance is depends on where you are. In the
Netherlands and Scandinavian countries the incidence of MRSA in hospitals is low
(<1%) [26] thanks to a “seek and destroy” policy, while in the USA and UK the
incidence of MRSA is much higher at around 42% [27]. This situation may well change
with the increase in community acquired resistance and increasing numbers of species
developing resistance. Bacteriophage could be an answer to the problem as they have a
totally different killing mechanism to antibiotics and they will evolve to meet whatever
changes occur in bacteria. On the negative side bacterial resistance to bacteriophages
does arise with a similar frequency to that of antibiotic resistance [28] which will incur a
cost in isolating appropriate new variants, which may not be as easy as sometimes
assumed, and cost in regulatory approval for the new strains used.
Advantage three: “Bacteriophages are safe and cheap and easy to produce”
The limited trials evidence, animal studies and anecdotal evidence all agree that
bacteriophages are “low risk” although lysogenic bacteriophages, ones with toxin or
virulence sequences or genetically engineered bacteriophages will be scrutinised more
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Bacteriophage therapy
closely by regulators. The likelihood is that regulatory requirement can be readily met,
but the evidence will have to be produced with GLP data. Exactly how regulators will
view bacteriophages, given the impossibility of a “species” definition, is not clear; a
single bacteriophage or cocktail of bacteriophages could be defined on the basis of, say,
gene sequence, but given the cost of the regulatory process, the necessity of changing the
bacteriophages regularly to counteract bacterial resistance and the limited market that
may exist for bacteriophage therapeutic products, such a limited approval could stop the
commercial development of bacteriophage therapeutics. The cost of bringing a typical
antibiotic to market is estimated at between $400 to $800 million [29] most of that being
regulatory, particularly the phase three clinical trial. Costs for a bacteriophage therapeutic
are unlikely to be less for clinical trials.
Efficacy of external bacteriophage treatments:
Wounds
Markoishvili et al [30] showed that bacteriophages soaked into a biodegradable film
healed ulcers in 70% of 96 patients. These ulcers had not been healed by conventional
treatment. Microbiological assessment was only available from 22 of the patients where
healing was associated with elimination of the pathogen from the wound. The dressing
also contained the antibiotic ciprofloxacin and bacteriophages commercially known as
‘PyoPhage’ active against strains of Pseudomonas aeruginosa, Escherichia coli,
Staphylococcus aureus, Streptococcus and Proteus. The healing of these wounds could
not be solely attributed to the bacteriophage.
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Bacteriophage therapy
Soothill [31] reported that skin grafts infected by P. aeruginosa consistently failed
in a guinea pig model. When these infected grafts were treated with 106 pfu of specific
bacteriophage, 6 out of 7 grafts took. Soothill and colleagues also demonstrated efficacy
with a bacteriophage against S. aureus in a rabbit abscess model [32]. The sewerage
derived bacteriophage reduced the abscess area and the count of S. aureus in the abscess
in a bacteriophage-dose dependant way.
Recent studies have reported that 87% of mice administered with a lethal dose of P.
aeruginosa in a mouse burn model were protected by a single dose of bacteriophage
cocktail injected intraperitoneally [33].
Capparelli et al [34] showed that bacteriophage against S. aureus could prevent abscess
formation and reduce the bacterial load and weight of abscesses when S. aureus was
injected into the flanks of mice.
Gut
A study by Smith et al [35] demonstrated that bacteriophage could cure severe diarrhoea
caused by an enteropathogenic E. coli (EPEC) strain in calves. The single dose of
bacteriophage was given at the same as the bacteria. Other cows that were feeding on
litter contaminated by these bacteriophage infected cows were protected against the
diarrhoea as well.
This study showed the importance of matching the bacteriophage against the EPEC strain
inoculated, there was also the presence of bacteriophages- resistant EPEC strains which
were as virulent as the original strain.
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Bacteriophage therapy
Recently Watanabe et al [36] showed that oral administration of bacteriophages was
effective in 66.7% of mice in a murine model of gut derived sepsis caused by P.
aeruginosa.
Ears
Marza et al [37] reported treatment of a dog with chronic bilateral otitis external that had
consistently grown P. aeruginosa. This infection had failed to be resolved after repeated
courses of topical and systemic antibiotics. After inoculation with 400pfu of
bacteriophage into the auditory canal there was marked improvement in the clinical signs,
27 hr after treatment.
At a recent conference it was revealed that a human trial of a bacteriophage cocktail
against P. aeruginosa has been successful in treating patients with chronic ear infections.
The trial was carried out in a specialised central London hospital and was carefully
devised with a double blind study and finished in November 2007; publications are in
preparation (www.biocontrol-ltd.com).
Internal Bacteriophage therapy:
The treatment of systemic infections is the most challenging environment for
bacteriophage action, with issues of compartmentation, host defense and kinetics, which
are not present to the same degree in topical applications, creating real or imaginary
barriers.
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Bacteriophage therapy
There are three aspects to consider when evaluating bacteriophage therapy; the
comparison with existing antibiotic treatments, the efficacy of bacteriophage treatment
and the cost to benefit of the treatment.
Trials of bacteriophage therapy in humans has been limited to studies in Eastern Europe
and Russia. Reviews of the Soviet literature [38, 39, 14] indicate a high level of success
for various forms of bacteriophage therapy, mostly oral bacteriophage delivery for
dysentery or local treatment of wounds. Slopek et al [40] reported bacteriophage
treatment of septacaemia with a 90% success rate but such trials have not been
sufficiently rigorous to be of regulatory usefulness. What is clear from such work is that
bacteriophage therapy has potential in the treatment of acute or chronic infections but
well designed trials are needed to validate this claim.
Animal trials of injected bacteriophages have been carried out in a number of species
with encouraging results. Intramuscular injection of lytic bacteriophages was used to treat
experimental E. coli septicemia in chickens and calves [41]. Chickens given equal doses
of bacteriophage and bacteria showed no morbidity, significant protection was shown by
1% bacteriophage/ bacteria doses, indicating bacteriophage multiplication in vivo. With
calves, intramuscular injection of bacteriophages delayed the onset of an experimental
infection of E. coli and increased survival times.
Mouse models of bacterial infection have had some success; a significant study on
vancomycin-resistant Enterococcus faecium (VRE) infection [42] demonstrated that
intraperitoneal administration of bacteriophages isolated from sewage against VRE- E
faecium prevented death.
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Bacteriophage therapy
When given 45 minutes after infection the bacteriophages prevented bacteraemia with a
reduction in circulating bacteria. Fully effective treatment was observed up to five hours
after infection, but was less effective beyond this time. The kinetics of bacteriophages in
model situations has been examined [43,44]; the outcome of treatment with a self
replicating agent such as bacteriophages was shown to depend critically on the initial
numbers and replication rates of both the bacteriophages and their bacterial hosts.
Descriptions of their interactions follow a predator – prey relationship rather than
classical pharmacokinetics.
Smith and Huggins [45] showed that bacteriophage lytic for an E. coli strain
(O18:K1:H7) which causes generalised infections in both man and animals was more
effective than conventional antibiotics in a mouse model. The E. coli strain was injected
intracerebrally into groups of 32 mice. At the same time the mice were either injected
with a single dose of anti-K1 bacteriophage intramuscularly or by multiple intramuscular
doses of antibiotics (streptomycin, tetracycline, ampicillin, chloramphenicol or
trimethoprim-sulfafurazole. In controls (no, bacteriophage or antibiotics), 31 out of 32
mice died, The lowest death rate noticed was in the bacteriophage treated group, in which
13 mice died. The antibiotic treated mice fared worse with 18 mice dying in the
streptomycin treated group and 26-28 dying in all the other antibiotic treated groups.
Further observations were that bacteriophage resistant strains of E. coli were isolated
from the bacteriophage treated group. This E. coli strain did not produce any K1 capsule,
according to the failure to precipitate anti-K1 antiserum on agar. Previous studies have
shown than K1 mutants had greatly reduced virulence when injected into mice or
chickens due to their inability to invade tissues [46].
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Bacteriophage therapy
Soothill [47] reported the protective ability of bacteriophages lytic against strains of
Acinetobacter baumanii, P. aeruginosa and S. aureus in experimental infections of mice
to investigate their potential for the treatment of infections of man. As few as 102 particles
of an Acinetobacter bacteriophage protected mice against five times the LD50 (1 x 108
cfu) of a virulent strain of A. baumanii, and the bacteriophages multiplied in the mice. A
Pseudomonas bacteriophage protected mice against five times the LD50 of a virulent
strain of P. aeruginosa, with a PD50 of 1.2 x 107particles. A Staphylococcal
bacteriophage failed to protect mice infected with a strain of S. aureus. Another failure
was reported by Skurnik [11].
Capparelli et al [34] reported that that 109 pfu of bacteriophage against S. aureus was
protective against a lethal dose of S. aureus in 97% of mice when administered with the
bacteria. The group also reported that mice infected intravenously with the lowest dose of
S. aureus strain not cleared by the innate immune system could be cleared of the bacterial
infection by giving the mice a single dose of bacteriophage (109 pfu) 10 days after initial
bacterial infection.
Conclusions.
These studies support the view that bacteriophages could be useful in the treatment of
human infections particularly those caused by antibiotic-resistant strains of bacteria but
that they are not a “miracle cure”. Assuming that the efficacy of bacteriophage treatment
is similar to that of antibiotics (and it is worth remembering that antibiotics are not 100%
effective even with sensitive bacteria!) then the availability of bacteriophage therapy will
depend on sensible and appropriate regulation and realistic business plans.
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Bacteriophage therapy
Acknowledgements:
The authors would like to thank Fiona Holland for some material used in this review.
Potential conflict of interest:
Both authors are associated (MM) or employed (JS) by Blaze v-t. This company uses
bacteriophages non clinically and is not involved in bacteriophages therapy, so no
conflict of interest is perceived.
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