1. health and fitness
2. therapy

To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
CHAPTER 112
Phage Therapy: Bacteriophages as Natural,
Self-Limiting Antibiotics
Elizabeth Kutter, PhD; Sarah Kuhl, MD, PhD; Zemphira Alavidze, PhD; and Bob Blasdel, BS
INTRODUCTION
Phage therapy involves the use of bacteriophages—viruses that only attack bacteria
and are very host specific —to kill pathogenic microorganisms. The art was first
developed at the Pasteur Institute in Paris early in the twentieth century, but since
the advent of chemical antibiotics in the 1940s, it has been little used in the West.
Today, however, the increased prevalence of bacteria that are resistant to most or all
available antibiotics is precipitating a major health crisis, as was again passionately
stressed by the World Health Organization in their call to action on World Health
Day, 2011.1 Here, we summarize the evidence that the available data strongly support expedited evaluation of phage therapy to help address this serious menace.2-14
Extensively documented results of French and Eastern European therapeutic
phage applications are encouraging, but most involved individualized applications
to infections recalcitrant to all other available treatments rather than double-blind
clinical trials. There have also been encouraging recent developments. In 2006, the
U.S. Food and Drug Administration (FDA) and the European Union (EU) both
approved phage preparations targeting Listeria monocytogenes on ready-to-eat foods.
The Nestlé Corporation is currently carrying out an extensive project in Dhaka,
Bangladesh, to study the safety and efficacy of phage therapy in treating enterotoxigenic Escherichia coli and enteropathogenic E. coli induced diarrhea in children.15
Either of two preparations, a novel cocktail of T4-like phages used in earlier safety
trials as well as a commercially available Russian anti-E. coli phage cocktail (Microgen), or a placebo is being added to the standard oral rehydration solution currently
in use in double-blind fashion. This work demonstrates all the key elements of modern clinical trials. The phages being used for the main experimental arm of the trial
were isolated from the stools of pediatric diarrhea patients in Bangladesh, with the
broad-spectrum ones applicable for phage therapy all turning out to be members of
the highly studied T4 family (see the following).16 They have also reported the details
of their very extensive genomic, mouse and human safety studies of representative
phages in their set.17
This chapter was written to put phage therapy into historical and ecologic perspective and to explore very interesting early research in France, the United States, and
Eastern Europe, as well as growing recent studies worldwide, with the hope that this
modality will soon be available for external applications while researchers deal with the
challenges of getting funding for full-scale clinical trials of more invasive approaches.
CHAPTER CONTENTS
Introduction, 1
Historical Context, 1
Discovery and Early Research, 1
Initial Attempts at Commercialization, 3
Specific Problems of Early Phage Therapy Work, 3
Properties of Bacteriophages, 3
Obligatorily Lytic Phages and the Development of
Molecular Biology, 4
Phage Interactions in the Body, 5
Clinical Application, 6
Clinical Research at the Institute of Immunology and
Experimental Medicine, Polish Academy of
Sciences, 7
Clinical Research at the Bacteriophage Institute,
Tbilisi, 7
Staphylococcus Aureus Infections, Whether or Not
Methicillin-Resistant, 8
Urogenital Tract Infections, 9
Gastrointestinal Infections, 9
Respiratory Tract Infections, 9
Ear Infections, 9
Toxicology, 9
Drug Interactions, 10
Dosing Strategy, 10
Conclusion, 10
Acknowledgments, 10
HISTORICAL CONTEXT
Discovery and Early Research
Edward Twort and Felix d’Herelle independently reported the isolation of filterable
entities capable of lysing bacterial cultures and of producing multiple small cleared
areas on bacterial cultures when diluted, rather than showing a minimum inhibitory concentration, implying that discrete particles were involved. They are jointly
given credit for the discovery of bacteriophages. It was d’Herelle, a Canadian working at the Pasteur Institute in Paris, who gave the name bacteriophage to these entities he discovered in the stools of soldiers recovering from dysentery—using the
suffix phage “not in its strict sense of to eat, but in that of developing at the expense
of.”18 He soon also isolated phages targeting avian typhosis and tested their application during an outbreak in French chickens, showing that three quarters of the
112-1
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
112-2
SECTION 5
|
PHARMACOLOGY OF NATURAL MEDICINES
untreated chickens died, but all of the infected chickens treated
with phages survived. He then accepted a request to treat several
children battling dysentery at the Paris Hospital des EnfantesMalades; the day before treating the first child, he and several
assistants swallowed far more phages than they would be administering, thus carrying out the first known Phase I clinical trial. This
trial was fully successful, but d’Herelle turned to intense study of
phages before publishing the results or carrying out further therapeutic applications. Thus, the first-known publication discussing
successful phage therapy was published in 1921 from Belgians
Bruynoghe and Maisin,19 who used phages to treat staphylococcal
skin infections.
In a 300-page 1922 book, The Bacteriophage: Its Role in Immunity,18 d’Herelle wrote the original, classic descriptions of plaque
formation and composition, infective centers, the lysis process,
host specificity of adsorption and multiplication, the dependence
of phage production on the precise state of the host, isolation of
phages from various sources, and the factors controlling stability of
the free phage. He quickly became fascinated with the apparent
role of phages in the natural control of microbial infections, and
noted the frequent specificities of the phages isolated from recuperating patients for disease organisms infecting them and the rather
rapid variations over time of the phage populations. Throughout
his life, he worked to develop the therapeutic potential of properly
selected phages against the most devastating health problems of the
day, traveling to many parts of the world, teaching at Yale from
1928 to 1933, and establishing his own Laboratoire du Bactériophage in Paris, run by his son-in-law, Theodore Mazure, which
produced the first commercial phage cocktails—Bacté-Coli-Phage,
Bacté-Intesti-Phage, Bacté-Dysentérie-Phage, Bacté-Pyo-Phage,
and Bacté-Rhino-Phage. Although France is clearly a western
country, most of the literature reviews of phage therapy have
ignored the successful continuation of phage therapy in France
until the early 1990s, which largely used well-made phage preparations produced by d’Herelle’s laboratory or by the Bacteriophage
Service of the Lyon and Paris branches of the Institut Pasteur.
Key to d’Herelle’s many successes was that he focused intensely
on understanding phage biology and on applying that knowledge in
his production and application of phages, including careful ongoing quality control, close work with physicians, and development of
appropriate treatment modalities. He wrote several more detailed
books on phage and phage therapy, two of which were also translated into English (1926 and 1930). The depth of his insights into
the practice of phage therapy became more accessible via the recent
publication of a translation of the appendix of his book.18 Through
much of the time, he worked closely with George Eliava, director of
the Georgian Institute of Microbiology, who had seen bacteriocidal
action of the water of the Koura River in Tbilisi (Tiflis) that he
could not explain until he became familiar with d’Herelle’s work
while spending 1920 to 1921 at the Pasteur Institute.18 Over the
years, the two developed plans to found an Institute of Bacteriophage Research in Tbilisi as a world center of phage therapy, including scientific and industrial facilities and supplied with its own
experimental clinics. A large campus on the river Mtkvari was allotted for the project in 1926. D’Herelle sent supplies, equipment,
and library materials. In 1934 to 1935, he visited Tbilisi for
6 months, set up his laboratory, and wrote a book, The Bacteriophage and the Phenomenon of Recovery,20 which was translated into
Russian by Eliava and had a very strong impact on Stalin, and thus
on the implementation of phage therapy in the Soviet Union.
D’Herelle intended to move to Georgia; his cottage still stands on
the institute’s grounds. However, in 1937, Eliava was arrested as a
“people’s enemy” by Beria, then head of the Georgian NKVD and
soon to direct the Soviet NKVD as Stalin’s much-feared henchman.
Eliava was executed, sharing the tragic fate of many Georgian and
Russian progressive intellectuals of the time, and d’Herelle, disillusioned, never returned to Georgia. However, their Bacteriophage
Institute survived under the leadership of a group of women well
trained by Eliava and d’Herelle. The Institute was put under the
People’s Commissary of Health of Georgia in 1938 and was transferred to the All-Union Ministry of Health in 1951, taking on the
leadership role in providing bacteriophages for therapy and bacterial typing throughout the Soviet Union. Hundreds of thousands of
pathogenic bacterial samples were sent to the Institute from
throughout the Soviet Union to isolate and produce more effective
phage strains and to better characterize their usefulness. A new fivestory building provided facilities for making two tons of phage
products two or more times a week, with 80% of the product going
to the Soviet military. These included tablets against dysentery, pyophage cocktails targeting wound and other purulent infections, as
well as intestiphage intended for a wide range of enteric pathogens.
Much of the main building held research laboratories continually
isolating new phages, upgrading therapeutic cocktails, working on
anaerobes and defenses against potential biowarfare, and collaborating closely with military and civilian physicians to test the phage
preparations and ways of administering them. These were the
golden years of the Institute, when its employees were among the
best paid in Tbilisi, and there were no shortages of supplies, new
research targets, or potential customers. In 1988, an official Scientific Industrial Union “Bacteriophage” was formed, centered in
Tbilisi with branches in Ufa, Habarovsk, and Gorki.
After the 1991 break-up of the Soviet Union, the Russian sites
were gradually pulled into the developing pharmaceutical giant,
Microgen (eng.microgen.ru) whose products include a wide range
of phage preparations available in pharmacies, online, and in hospitals throughout the Soviet Union. What is now the Eliava Institute
lost most of its markets and funding and struggled for survival as the
factory portions were stripped away and privatized. Phages for Georgian use were made in 30-L batches packaged by hand in small
5-mL ampules, 10 to a box, and sold in the diagnostic clinic on the
Eliava campus in laboratories that had once focused on researchscale preparations, and where laboratory leaders did the actual testing and diagnosis, supporting the laboratories while continuing to
acquire all the newest pathogenic strains for research and production
purposes. Often, the power and/or gas went out, and no new young
people were being trained. By 1996, help became available from the
International Science and Technology Centers and the Civilian
Research and Development Fund, set up by North Atlantic Treaty
Organization (NATO) and the United States, the EU, the Phage
Biotics Foundation, and other sources to fund civilian research by
scientists formerly supported by the military. With hard work and
outside help, the institute is again thriving at its original site on the
Mtkvari. Their work is further discussed in the following.
Initial Attempts at Commercialization
Phage therapy has been explored extensively, with successes being
reported for a variety of diseases, including dysentery, typhoid and
paratyphoid fevers, pyogenic and urinary tract infections, and
cholera.4 Phages have been given orally, through colon infusion,
and as aerosols as well as infused directly into lesions. They have
also been given as injections: intradermal, intramuscular, intraduodenal, intraperitoneal, into the pericardium and arteries leading to infected areas, as well as intravenously. The early strong
interest in phage therapy is reflected in some 800 papers published
on the topic between 1917 and 1956. The reported results were
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
CHAPTER 112
|
Phage Therapy: Bacteriophages as Natural, Self-Limiting Antibiotics
quite variable. Many of the physicians, entrepreneurs, and pharmaceutical firms who initially became very excited by the potential clinical implications, especially in the pre-antibiotic era,
jumped into application efforts with very little understanding of
phages, microbiology in general, or the basic scientific process.
Thus, many of the studies were anecdotal and/or poorly controlled; many of the failures were predictable; and some of the
reported successes did not make sense in light of current knowledge. Too often, uncharacterized phages at unknown concentrations were given to patients without previous specific bacteriologic
diagnosis, and there is no mention of follow-up, controls, or placebos. Much of the understanding gained by d’Herelle was ignored
in this early work, and inappropriate methods of preparation,
“preservatives,” and storage procedures were often used. On one
occasion, d’Herelle reported testing 20 preparations from various
companies and finding that not one of them contained active
phages! Not surprisingly, a check of the product showed that one
phage had out-competed all the others, and this was not a polyvalent preparation. In general, there was little essential quality control except in a few research centers. Large clinical studies were
rare, and the results of those few that were carried out were largely
inaccessible outside of what was then the Soviet Union.
112-3
eukaryotic viruses, most phages have tails, the tips of which have
the ability to bind to specific molecules on the surfaces of their
target bacteria (Figure 112-2). The viral DNA is then ejected
through the tail into the host cell, where it directs the production
of progeny phages; often more than 100 are produced in just half
an hour from each bacterial cell.
Each strain of bacteria has characteristic protein, carbohydrate,
and lipopolysaccharide molecules present in very large quantities on
its surface. These molecules are involved in forming pores, motility,
and binding of the bacteria to particular surfaces. Each such molecule can act as a receptor for particular phages. Development of resistance to a particular phage generally reflects mutational alteration or
loss of its specific receptor; this loss frequently has negative effects on
the bacterium, often making such mutants less virulent, and does
not confer protection against the many other phages that use different receptors. Each kind of bacterium has its own phages, which can
generally be isolated wherever that bacterium grows: from sewage,
feces, soil, even ocean depths and hot springs, but finding phages
suitable for therapeutic applications is much easier for some species
than for others; this relates significantly to the ability to grow the
host in the laboratory. The process of isolation is straightforward for
phages targeting many of our best-studied pathogens. An environmental sample is combined with an appropriate nutrient-fortified
Specific Problems of Early Phage Therapy Work
Some still believe (erroneously) that phage therapy was proven not
to work; however, it simply was never adequately researched for a
variety of reasons, and the work that was done well is not widely
enough known. It is thus important to carefully consider the reasons
for the early problems and for the questioning of efficacy:
• Paucity of understanding of the heterogeneity and ecology of
the phages and the bacteria involved
• Lack of availability or reliability of bacterial laboratories for
carefully identifying the pathogens involved (important considering the relative specificity of phage therapy)
• Use of too few phages in infections that involved mixtures of
different bacterial species and strains
• Failure to select obligatorily lytic phages against the target bacteria before using them in patients
• Emergence of phage-resistant bacterial strains, through selection of resistant mutants (especially if only one phage strain was
used against a particular bacterium) or through lysogenization
(if temperate phages were used, as discussed later)
• Failure to appropriately characterize or titer phage preparations,
many of which, even from major companies, were shown to be
totally inactive.21,21a
• Failure to neutralize gastric pH before oral phage administration
• Inactivation of phages by both specific and nonspecific factors
in bodily fluids
All of these factors need to be taken into consideration as we now
work to formally document phage efficacy and integrate phages
into medical practice worldwide.
FIGURE 112-1 Phage diagram (bacteriophage T4).
PROPERTIES OF BACTERIOPHAGES
Viral particles are like spaceships that are able to transfer their
genomes between susceptible cells where they can reproduce. In
the case of bacteriophages, the targets are specific kinds of bacterial
cells, specific to varying degrees for each phage; they cannot infect
the cells of more complex organisms. Each virus consists of a long
nucleic acid polymer (DNA or RNA) containing the genetic information that determines all of the properties of the virus, which is
carried around packaged in a protein coat (Figure 112-1). Unlike
FIGURE 112-2 Electron micrograph of phage infecting a bacterium.
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
112-4
SECTION 5
|
PHARMACOLOGY OF NATURAL MEDICINES
solution and several targeted bacteria, incubated over night, and
10 μL each of a series of 10-fold dilutions spotted on a nutrient-agar
plate prepared with a single strain of target bacteria. The next day, a
dense bacterial lawn is seen, hopefully with cleared spots and, at
some dilution, dotted with small, cleared plaques. Each plaque contains about a billion phages, all of them progeny of a single initial
phage. Individual plaques are plucked and cultured to give a homogeneous stock of that particular phage, whose host ranges and other
properties can then be studied.
One major source of confusion in the early phage work was that
new phages were often isolated by each laboratory, so there was
little continuity or basis for comparison. Key technical developments in the 1930s to 1940s that helped clarify the general nature
and properties of bacteriophages were (1) the concentration and
purification of some large phages by means of very high-speed
centrifugation and the demonstration that they contained equal
amounts of DNA and protein; and (2) visualization of phages by
the electron microscope (EM).22,23 Phages specific for virtually
every well-studied bacterial species have now been isolated, and
increasingly, many are becoming well classified.
This EM work also helped resolve the major disputed question as
to whether the lytic principle termed bacteriophage simply reflected
an inherent property of the specific bacteria or required regular
reinfection by an external agent, as claimed by d’Herelle. During
the 1930s and 1940s, it gradually became clear that in some senses
both were true—that there were two quite fundamentally different
groups of bacteriophages. Obligatorily lytic phages always have to
infect from outside, reprogram the host cell, and release a burst of
phage through breaking open, or lysing, the cell after a relatively
fixed short interval. Temperate phages, in contrast, have another
option; they can actually integrate their DNA into the host DNA,
much as HIV can integrate the DNA copy of its RNA. For several
reasons, such temperate phages are not appropriate for therapeutic
applications. They generally make the host resistant to related
phages, blocking treatment efficacy, and also may carry genes that
actually increase the pathogenicity of the host; very specific prophages are a key factor in such diseases as cholera and diptheria.
Using the EM, each phage family was found to have its own
specific shape and size, from “lunar lander”–style complexity with
a contractile tail and long tail fibers attached to a baseplate, to
globular heads with long or short tails, to the small filamentous
phages that looked much like bacterial pili (Figure 112-3). With
recent advances in DNA sequencing techniques, our understanding of various phages is exploding in powerful and important
ways; the genomes of several hundred phages, infecting a variety
of organisms, have now been sequenced, revealing a remarkable
variety of types and properties that can be very important when
considering potential therapeutic applications. In general appearance, 95% of the studied phages belong to one of the three general
tailed morphotypes: the short-tailed podoviridae, the siphoviridae
with long, often flexible tails; and the myoviridae, with tails composed of an inner tube and an outer contractile sheath attached to
complex baseplate. There are both obligatorily lytic and temperate
phage families with each of these three general morphotypes, so
other kinds of data are also needed to tell whether a newly isolated
phage is temperate or lytic.
Obligatorily Lytic Phages and the Development
of Molecular Biology
A much better understanding of the interactions between the lytic
lifecycle of phages and bacteria began with one-step growth curve
experiments. These demonstrated an eclipse period, during which
P
lp
M
S
Mi
C
T
L
PI
Cy
SSVI
II
LI
FIGURE 112-3 Various phages.
the DNA began replicating and there were no free phages in the cell;
a latent period of accumulation of intracellular phages; and a precisely timed lysis process that released the phage to go in search of
new hosts. This phage infection cycle is illustrated in Figure 112-4
for coliphagephage T4, which does a particularly effective job of
shutting off all host functions and whose family is very widespread
in nature and in therapeutic phage preparations for enteric bacteria.
In 1943, an event occurred that had a major impact on the
orientation of phage research in the United States and much of
western Europe, strongly shifting the emphasis from practical
applications to basic science. Physicist-turned–phage biologist
Max Delbruck met with Alfred Hershey and Salvador Luria to
form the “Phage Group” and establish the long-time annual phage
course and meeting at Cold Spring Harbor, Long Island. A major
element of the success of phages as model systems for working out
fundamental biologic principles at the molecular level was that
Delbruck persuaded most U.S. phage biologists to focus on one
bacterial host (E. coli B) and seven of its highly lytic phages, arbitrarily named types T1 through T7. Fortuitously, T2, T4, and T6
are quite similar to one another, defining a family of myoviridae
now called the T-even phages, which were key in demonstrating
that DNA is the genetic material, that viruses can encode enzymes,
that gene expression is mediated through special “messenger
RNA,” that the genetic code is triplet in nature, and other fundamental concepts. The negative side of this focus on a few phages
growing on one host under rich laboratory conditions was that
there was very little study or awareness of the ranges, roles, and
properties of bacteriophages in the natural environment, or of
potential applications. On the positive side, most of the strongly
lytic phages selected for therapeutic applications targeting enteric
phages have turned out to belong to one or another of these three
very well-studied families of phages (as confirmed by sequencing
data as well as morphologic details), which is very useful as we
work to assure their safety and to understand the physiologic
properties involved.
The temperate phages generally encode repressors to turn off
most of their own genes when they are in the temperate pro-phage
state, integrases to let them insert themselves into the DNA of
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
|
CHAPTER 112
Phage Therapy: Bacteriophages as Natural, Self-Limiting Antibiotics
112-5
Head
precursors
DNA
Replication
DNA
Late
mRNAs
Early
mRNAs
Early
proteins
DNA
precursors
Late
proteins
Nucleases
Host
chromosome
0
5
Membrane
components
10
15
20
25
Minutes after Infection
FIGURE 112-4 Bacteriophage intracellular growth cycle. Noteworthy features: both nucleolytic action on host chromosome and new phage enzymes furnish DNA precursors; replicating DNA is much longer than virion DNA; a number
of phage-coded proteins become associated with the host membrane to alter host functions and to facilitate phage
assembly and maturation.
their hosts, and excision enzymes to cut their DNA back out to
enter a lytic cycle. Obligately lytic phages generally have more
extensive mechanisms for shutting off the host, often involving
nucleases and transcription factors, as well as a variety of small
proteins made under the control of strong promoters very early in
infection to re-direct cellular metabolism; T4, for example,
encodes at least 50 such proteins.
PHAGE INTERACTIONS IN THE BODY
A number of early experiments involving phage injection into animals led to widespread belief that phage therapy could not succeed
because the phages were too rapidly cleared by the immune system. Early experiments in rabbits, rats, and mice showed rapid
disappearance of phage from the blood and organs, but long-term
survival in the spleen.24,25 However, these experiments were done
in the absence of host bacteria in which the phage could multiply
and were carried out by the unnatural mode of intravenous injection, exposing the phage almost immediately to the reticuloendothelial system. Many later studies made it clear that phages are
seen in the mammalian circulatory system for prolonged periods
when they are entering it from some sort of reservoir in other tissues, and the host is dealing with infection by a bacterium in
which they can replicate —precisely the sort of situation seen in
phage therapy as currently practiced.
This pattern is particularly clear in research published in 1943
by noted Harvard bacteriologist René Dubos26 (Figure 112-5).
Dubos et al26 injected mice intracerebrally with a dose of a smooth
Shigella dysenteriae strain that was sufficient to kill more than 95%
of the mice in 2 to 5 days and treated them with intraperitoneal
injection of a mixture of phages that had been isolated from New
York City sewage, grown in the same bacteria, and purified only
by sterile filtration. With no treatment or when treated with filtrates of bacterial cultures or with heat-killed phage, only 3 of 84
mice (3.6%) survived; in contrast, 46 of 64 (72%) of the mice
given 107 to 109 phages survived. Pharmacokinetic studies on the
mice showed that when phages were given to uninfected mice,
they appeared in the bloodstream almost immediately, but the levels started to drop within hours and very few were seen in the
brain, as shown in Figure 112-5. However, in infected animals,
brain levels of viable phage appropriate to those bacteria soon far
exceeded blood levels; around 107 to 109 phages/g were often seen
between 8 and 114 hours after administration, with the level
dropping between 75 and 138 hours after phage addition. After
the first 18 hours, blood levels were far lower than brain levels, but
phages were still present in blood at 104 to 105 phages/mL in those
animals in which the brain levels were still high.
Equally well-controlled experiments performedby Henry Morton and Enrique Perez-Otero from 1943 to 1945 at the University of Pennsylvania supported those of Dubos et al26 and further
showed the lack of any protection when phages with inappropriate host specificities were used. These results clearly established
that (1) the phages themselves were responsible, not something
in the lysate that just stimulated normal immune mechanisms;
(2) phages could rapidly find and multiply in foci of infection
anywhere in the body, including crossing the blood–brain barrier; and (3) phages could be maintained in the circulation as
long as there was a privileged reservoir of infection where phages
were continually being produced. A final review authorized by
the Council on Pharmacy and Chemistry discussed the major
advantages of phages, such as their ability to replicate into problem areas and treat localized infections that are relatively inaccessible via the circulatory system, and the fact that their high
specificity greatly aided in reducing later resistance problems.27
The review also emphasized that most of the earlier phage research
had been so poorly conceived and/or carried out that it offered
no proof either for or against the promise of phages as antibiotics; therefore, the negative conclusions of the earlier American
Medical Association reviews were neither unexpected nor very
relevant to the potential for eventual success.
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
112-6
SECTION 5
|
PHARMACOLOGY OF NATURAL MEDICINES
1010
109
Infected brain
108
PFUmL–1
107
106
Infected blood
105
104
103
Normal blood
Normal brain
102
10
20
30
40
50
60
70
80
90
100
110
120
130 140
Time (hr)
FIGURE 112-5 This figure, based on the data in the 1943 mouse studies of Dubos et al,26 provides significant
insight into why phage therapy works well even in treating infections that antibiotics cannot treat. When the mice
were injected intraperitoneally with 109 phage, they quickly appeared in the bloodstream and some even crossed the
blood−brain barrier, but they were rapidly cleared. However, when the mice were also injected intracerebrally with
Shigella dysenteriae, the host for these phages, 46 of 64 of the mice survived (compared with 3 of 84 in the absence
of appropriate viable phage), and the brain level of phage climbed to over 109/g. Once the bacteria cleared, the level
of phage dropped below detection limits.
The context of these studies sheds enlightening insights into the
historical course of phage therapy in the United States.28 In 1942,
both The Lancet and the British Medical Journal published editorials about the apparently successful use of antidysentery phages by
the Soviet military in the Middle and Far East. By November
1942, the U.S. National Research Council Committee on Medical
Research began supporting research possibilities offered by antidysentary phages for dealing with this perpetual scourge of armies
(including the previous studies) in top U.S. bacteriology laboratories new to phage work, initially requiring them to keep the results
secret. This promising work ended in 1944, when the end of
World War II made penicillin generally available. The military
secrecy, the end of the war emergency funding, the rapid rise in
antibiotic availability and their broad-spectra “wonder-drug” status, and Max Delbruck’s success in persuading the phage community to shift its focus to basic molecular research involving a few
model systems, all contributed to the fact that there was little U.S.
follow-up to these interesting and successful results. Although the
results were published in major journals, few people even knew
about them, or about highly successful ongoing human applications, until they were recently rediscovered by Thomas Häusler.2
Penicillin works only against gram-positive bacteria, so it cannot treat typhoid fever, and early phage efforts against typhoid had
very mixed success. It was then found that the major pathogenic
strains of Salmonella typhi all carried one particular antigen,
named Vi (for “virulence”). In 1936, a pair of Canadians identified a number of phages specific against the Vi antigen; these are
still used as “typing phages” in rapidly identifying and following
outbreaks. In the late 1930s and early 1940s, physicians at the Los
Angeles County Hospital used phage treatments to help deal with
repeated serious outbreaks of typhoid that were killing one in five
of those afflicted.29 Walter Ward tested the Vi-specific phages
against mouse typhus and found that the death rate fell to
6% versus 93% in controls.29 When these phages were then used
to treat patients with typhoid, only 3 of 56 treated patients died
compared with the 20% mortality for the other treatments available at the time.30 Most impressively, this study reported that the
patients who received phage therapy rapidly changed from being
largely comatose to full of vigor, with renewed appetite, in 24 to
48 hours. In 1948 to 1949, near Montreal, Desranleau treated
nearly 100 patients with dysentery by giving them a cocktail of
six different Vi-specific phages, and the death rate dropped from
20% to 2%.30a By then, however, chloramphenicol had been
shown to work well against typhoid, and it was much easier for
pharmaceutical companies to deal with, so that seems to have
been the end of phage clinical trials in the Western hemisphere.
The high specificity of phages still plays a strong role in the
phage typing sets used for detecting and following problem strains
of such bacteria as Shigella, Salmonella, and E. coli, but phage
therapy itself is only beginning to stage a comeback.
CLINICAL APPLICATION
The growing understanding of phage biology has the potential to
facilitate more rational thinking about the therapeutic process and
the selection of therapeutic phages. However, there was generally
little interaction between those who were so effectively using phages
as tools to understand molecular biology and those working on
phage ecology and therapeutic applications. Many in the latter
group were spurred on by a concern about the rising incidence of
nosocomial infections and of bacteria resistance against most or all
known antibiotics, as well as by the fact that phages are far more
effective than antibiotics in areas of the body where circulation is
poor and does not disrupt normal flora. This strong sense of the
potential importance of phages was particularly seen in Poland,
France, Switzerland, and the former Soviet Union, where use of
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
CHAPTER 112
|
Phage Therapy: Bacteriophages as Natural, Self-Limiting Antibiotics
therapeutic phages never died out and where there has been ongoing research and clinical experience. In France, Dr. Jean-François
Vieu led the therapeutic phage efforts until his retirement 15 years
ago. He worked in the Service des Entérobactéries of the Pasteur
Institute in Paris and, for example, prepared Pseudomonas phages
on a case-by-case basis for patients. His experience there is discussed
in two articles.31,32 In Vevey, Switzerland, the small pharmaceutical
firm Saphal made “Coliphagine,” “Intestiphagine,” “Pyophagine,”
and “Staphagine” in drinkable and injectable forms, salves, and
sprays into the 1960s.28 The owner, Harrmann Glauser, had been
encouraged and trained by d’Herelle’s old colleague Paul Hauduroy,
who had become a professor of microbiology at the University of
Lausanne during the second World War. These phage preparations
were officially approved and were paid for by insurance.
Phage therapy was used extensively in many parts of eastern
Europe as a regular part of clinical practice, and companies in Russia
now make phages for this purpose. However, most of the research
and much of the phage preparation came under the direction of key
centers in Tbilisi, Georgia, and Wroclaw, Poland. In both cases, the
close interactions between research scientists and physicians played
an important role in the high degree of success obtained, just as was
the case for d’Herelle’s early work.
Clinical Research at the Institute of Immunology
and Experimental Medicine, Polish Academy of Sciences
Some of the most detailed publications documenting phage therapy are from the group led by Stefan Slopek, a long-time director
of the Wroclaw Institute of Immunology and Experimental Medicine, as well as his successors there. Slopek’s initial series of papers
described work completed in 1981-1986 with 550 patients in 10
Polish medical centers.33-35 The patients ranged in age from
1 week to 86 years and venues included the Wroclaw Medical
Academy Institute of Surgery Cardiosurgery Clinic, Children’s
Surgery Clinic and Orthopedic Clinic; the Institute of Internal
Diseases Nephrology Clinic; and the Clinic of Pulmonary Diseases. In 518 cases, phage use followed unsuccessful treatment
with all available methods, including antibiotics. The major categories of infections included long-persisting suppurative fistulas
and abscesses, septicemia, respiratory tract suppurative infections,
pneumonia, and purulent peritonitis. In a final summary article,
the results were carefully analyzed with regard to such factors as
nature and severity of the infection and monoinfection versus
infection with multiple bacteria.35 Rates of success ranged from
75% to 100% (92% overall), as measured by marked improvement in relevant physical condition, wound healing, and disappearance of titratable bacteria; 84% of subjects demonstrated full
elimination of the suppurative process and healing of local
wounds. Infants and children did particularly well. Not surprisingly, the poorest results occurred in elderly patients and those in
the final stages of extended serious illnesses.
Appropriate individual highly-virulent phages were selected from
their extensive collection. In the first study alone, 259 different
phages were tested (116 for Staphylococcus, 42 for Klebsiella, 11 for
Proteus, 39 for Escherichia, 30 for Shigella, 20 for Pseudomonas, and
1 for Salmonella); 40% of them were selected to be used directly for
therapy. All of the treatments were conducted in research mode,
with the phage prepared at the institute by standard methods and
tested for sterility. Treatment generally involved 10 mL of sterile
phage lysate given orally half an hour before each meal, with gastric
juices neutralized by ingesting (basic) Vichy water, baking soda, or
gelatin. In addition, phage-soaked compresses were generally
applied three times a day where dictated by localized infection.
112-7
Treatment ran for 1.5 to 14 weeks (mean, 5.3 weeks). For intestinal
problems, short treatment sufficed, whereas long-term use was necessary for such problems as pneumonia with pleural fistula and pyogenic arthritis. Bacterial levels and phage sensitivity were continually
monitored, and the phage(s) were changed if the bacteria lost their
sensitivity. Therapy was generally continued for 2 weeks beyond the
last positive test result for the bacteria. Few side effects were
observed; those that were seen seemed to be directly associated with
the therapeutic process.33
Various methods of administration were successfully used,
including oral, aerosols, and infusion, either rectally or in surgical
wounds. Intravenous administration was not recommended for
fear of possible toxic shock from bacterial debris in the lysates.33
However, it was clear that the phages readily entered the body
from the digestive tract and multiplied internally wherever appropriate bacteria were present, as measured by their presence in
blood and urine as well as by therapeutic effects.36 This interesting
and rather unexpected finding has been replicated in many studies
and systems.37-40
Detailed notes were kept throughout on each patient. The final
evaluating therapist also filled out a special inquiry form that was
sent to the Polish Academy of Science research team along with the
notes. The Computer Center at Wroclaw Technical University carried out extensive analyses of the data. These researchers used the
categories established in the World Health Organization’s (1977)
International Classification of Diseases in assessing results. They also
looked at the effects of age, severity of initial condition, type(s) of
bacteria involved, length of treatment, and other concomitant
treatments. After Slopek’s retirement, Dr. Beatta Weber-Dabrowska
carried on with the treatment work, publishing a summary in English of the results for the next 16 patients.41 In 1998, immunologist
A. Górski took over as Institute director and revived a strong focus
on phage work, with special emphasis on the immunologic consequences of phage treatment. These researchers are also now working
with the basic phage group of Dr. M. Lobocka in Warsaw to
sequence and further characterize key phages—an important step
in eventually making them available to the outside world.
Clinical Research at the Bacteriophage Institute, Tbilisi
Particularly extensive efforts for phage therapy were carried out
over many decades by scientists at the Bacteriophage Institute in
Tbilisi, Georgia, working closely with local physicians. Phage therapy is an accepted component of the general standard of care in
Georgia, used extensively in pediatric, burn, and surgical hospital
settings. Phage preparation was carried out on an enormous scale
before the breakup of the Soviet Union, employing 700 people in
the factory and several hundred more in the research arm of the
Institute, making 2 tons of phage products weekly to ship throughout the former Soviet Union. They were available both over the
counter and through physicians; 80% went to the military for
wound and burn infections and for preventing debilitating gastrointestinal epidemics. In hospitals, they were used to treat both primary and nosocomial infections, alone or in conjunction with
other antimicrobials. The International Science and Technology
Centers program, set up jointly by the United States, Europe, and
Japan to give constructive opportunities to scientists formerly
working with Soviet military projects, is now one of the strongest
supporters of basic and applied research in this area in Tbilisi.
From the Bacteriophage Institute’s inception, the industrial part
was run on a self-supporting basis, whereas its scientific branch was
government supported. The Institute carried out the extensive
studies needed for approval by the Ministry of Health in Moscow
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
112-8
SECTION 5
|
PHARMACOLOGY OF NATURAL MEDICINES
of each new strain, therapeutic cocktail, and means of delivery. This
careful study of the host range, lytic spectrum, cross resistance, and
other fundamental properties of the phages being used was a major
factor in the reported successes of their phage therapy work, as were
their methods for selecting highly virulent phages from among the
many available against any given host. Where necessary, new cocktails were prepared with broader host ranges. The depth and extent
of the work involved are impressive. For example, in 1983 to 1985,
the Institute’s Laboratory of Morphology and Biology of Bacteriophages carried out studies of growth, biochemical features, and
phage sensitivity on 2038 strains of Staphylococcus, 1128 of Streptococcus, 328 of Proteus, 373 of P. aeruginosa, and 622 of Clostridium
received from clinics and hospitals in towns across the former
Soviet Union. New broader acting phage strains were isolated and
were included in a reformulation of their extensively used Pyophage preparation. A good deal of work went into developing the
documentation for Ministry of Health approval of specialized new
delivery systems, such as a spray for use in respiratory tract infections, in treating the incision area before surgery, and in sanitation
of hospital problem areas. An enteric-coated pill was also developed, using phage strains that could survive the drying process, and
accounted for the bulk of the shipments to other parts of the former Soviet Union. Much work focused on combating nosocomial
infections, in which multidrug-resistant organisms have become a
particularly lethal problem. An exciting new product was licensed
in 2000 by the Georgian Ministry of Health. PhagoBioDerm is a
biodegradable, nontoxic polymer composite that allows the sustained release of the Pyophage cocktail of phages.42,43 One study
on PhagoBioDerm involved 107 patients with ulcers that had
failed to respond to conventional therapy—systemic antibiotics,
antibiotic-containing ointments, and various phlebotonic and vascular protecting agents. The ulcers were treated with PhagoBioDerm alone or in combination with other interventions during
1999 and 2000. The wounds or ulcers healed completely in 70% of
the 96 patients for whom there was follow-up data. In the 22 cases
for which complete microbiologic analyses were available, healing
was associated with the concomitant elimination or very marked
reduction of the pathogenic bacteria in the ulcers. A newly formulated version of PhagoBioDerm should soon be on the market; it is
much less expensive to produce and has other advantages.
Staphylococcus Aureus Infections,
Whether or Not Methicillin-Resistant
Methicillin-resistant S. Aureus (MRSA) is a particular concern
given its reduced susceptibility to antibiotic treatment, wide prevalence in hospital-acquired infections and in the community, and
potentially lethal and otherwise serious consequences. These pathogens are targeted by the anti-S. aureus activity of phage preparations
such as Pyophage (which include potent anti-Staphylococcus phages
of the broad-spectrum Sb1-staph phage K family; Kutter EM,
unpublished results). Here as elsewhere, there is no cross resistance
between phages and antibiotics. Furthermore, very little development of resistance to this family of phages has been observed, presumably implying that the still unidentified primary receptor is a
molecule of significant importance to the bacteria. Thus, so far as
phages are concerned, MRSA is simply another strain of Staphylococcus. Treatment of MRSA using phages can be accomplished by
local application for local infections or, if necessary, and with substantially more caution, more systemic dosing such as intraperitoneally for systemic infections.44 The use of phage treatment for
local infections, including particularly those due to Staphylococcus,
has the distinction of being one of only two phage therapy
strategies that were deemed to be convincingly efficacious by the
Eaton and Bayne-Jones report in 1934,45-47 and an otherwise phage
therapy skeptical publication.48 The first human phage therapy
publication reported on treatment of S. aureus skin infections.6,49,50
Phage preparations for systemic application were developed at the
Eliava Institute during the 1980s, including safety studies in human
volunteers without adverse effects. Phages were particularly effective in infants and in immune-compromised patients, and for infusion into the urethra in cases of pelvic inflammatory disease. The
preparation subsequently was used to treat 653 patients.6 Historically, questions have been raised as to whether the apparent efficacy
documented in these classic articles was due to the phage itself giving rise to bacterial lysis in situ. It was suggested that the debris in
the phage lysate, stimulating the host immune system, could be a
major factor in bacterial clearance.6 See Sulakvelidze and Barrow 51
and Kutter et al5 for discussion of what is known as Staph Phage
Lysate or simply SPL, produced by Delmont Laboratories. This
product is marketed as a veterinary vaccine conferring resistance to
staph through immune stimulation by its staphylococcal phage
induced bacterial lysis products, which is advertised as the major
active ingredient. It also contains viable phages, often at approximately 108 plaque forming units (PFU)/mL (Kutter EM, unpublished data; Kuhl SJ, unpublished data), a level as high as the total
phage in Pyophage as determined by direct fluorescent microscopic
count.51a These staph phage lysates initially were produced for
human as well as animal use against chronic infections.6 However,
in 1994, they were limited by the FDA to animal use pending further human efficacy trials, for which no funding has yet been found;
no questions have been raised as to their safety.
Phage use to prevent Staphylococcus infections has been both
proposed and employed. Phages have been used for disinfection in
Georgia to sanitize operating rooms and medical equipment and
prevent nosocomial infections.49 A complementary approach proposed by the company Novoltyics is to use “an aqueous suspension to treat nasal carriage of MRSA, thus significantly reducing
the incidence of MRSA transmission” (see www.novolytics.co.uk/
technology.html, see also Mann51b). O’Flaherty et al51c described
removal of S. aureus via experimental hand washing with a phagecontaining Ringers solution. Approximately 100-fold reductions
in bacterial densities were observed after washing with a solution
containing 108 phages/mL versus the phage-less control solution.
Leszczynski et al51d described the use of oral phage therapy for
targeting MRSA in a nurse who was a carrier. This individual had
MRSA colonized in her GI tract and also had a urinary tract infection. The result was complete elimination of culturable MRSA. In
an earlier publication, the same group argued that MRSA treatment using phages could be economically preferable to MRSA
treatment using antibiotics52; in contrast, see the discussion in
Abdul-Hassan, et al.53 Jikia et al53a described phage treatment of
MRSA infecting radiation burns (see further discussion following). Slopek et al35 reported 92.4% positive cases for phage treatment of 550 single- and mixed-etiology infections involving
S. aureus. Slopek et al34 specifically used phage treatment for suppurative staphylococcal infections, with a reported 93% “effective” rate “based on case history and data contained in a special
questionnaire,” while also using this treatment of various kinds of
Staphylococcus infections in children (95.5% positive results for
the 90 children treated).
Urogenital Tract Infections
Phages have been applied to treat various infections of the urogenital systems either systemically, via direct injection into the bladder
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
CHAPTER 112
|
Phage Therapy: Bacteriophages as Natural, Self-Limiting Antibiotics
or via topical application. Phage treatment of urogenital tract
infections could potentially be complimented by current naturopathic protocols involving alkalinization of the urine with citrates
and minerals. Eaton and Bayne-Jones45-47 in their 1934 report
were convinced of the efficacious use of phage therapy against cystitis. Letwiewicz et al54 described phage application rectally to target Enterococcus faecalis infection of the prostate, with substantial
success in eliminating the target bacteria. In this case, the phages
were presumed to be taken up through the rectal wall. Letarov
et al55 noted that rectal phage suppositories are available on the
Russian market. Slopek et al35 reported 92.9% positive results for
phage treatment of 42 “diseases of the genitourinary tract.”
Chanishvili’s and Sharp’s56 book has chapters on “Phage Therapy
in Urology” and “Phage Therapy in Gynecology.” They summarize
the results there as follows: “In cases of acute cystitis a therapeutic
effect was observed within 4–5 hours of the first administration
and resulted in relief of pain, a decrease in the frequency of urination and a normalization of the composition of the urine. Full
recovery was achieved within 1–3 days in all 13 cases (100%) however treatment of chronic forms of cystitis was less successful, with
only a moderate improvement observed.” Supported by a 3-year
grant from the International Science and Technology Centers, the
Eliava Institute developed a new phage cocktail specifically targeted
against a large pool of bacteria from prostatitis and urinary tract
infections. At the 2009 Evergreen International Phage Biology
Meeting, Alavidze reported on its production and on very successful preliminary trials involving over 100 patients. A western-style
double blind, placebo-controlled trial involving addition of this
phage to the standard treatment at one of the major clinics in
Tbilisi is currently in the planning stage.56a
Gastrointestinal Infections
From the beginning, acute diarrheal infections were a major and
very successful part of phage therapy practice, as discussed previously, and the major clinical trial currently under way involves
work by Nestle in Bangladesh to stem the death toll caused by
infant diarrhea. However, most GI problems, such as irritable
bowel syndrome, diverticulitis, and Crohn’s disease, may involve
long-term chronic infections and immune system challenges.
Although there is no body of literature to build on from Tbilisi or
from Poland on the latter application, this would appear to be an
area well worth exploration.
Respiratory Tract Infections
Respiratory infections can be differentiated into numerous types;
however, phage therapy is limited in efficacy to those with a bacterial
etiology. Weber-Dabrowska et al57 reported successful treatment of
pneumonia in six cancer patients. Similarly, Slopek et al58 reported
86.7% positive results for phage treatment in 180 “diseases of the
respiratory system” (see also Slopek et al33). The first case studies of
phages used to treat people for chronic infections of S. aureus and
P. aeruginosa in Tbilisi were recently published; these studies used
Pyophage and a fully sequenced S. aureus phage delivered by standard cystic fibrosis nebulizers.59,60 The latter study included a
detailed description of the successful treatment of P. aeruginosa
infection in the lungs of a 7-year-old patient (using Pyophage) along
with treatment of a S. aureus co-infection in the same patient using
phage Sb-1. The company previously known as Biocontrol reported
an interest in expanding its anti-Pseudomonas phage therapy efforts
to include treatment of children with cystic fibrosis.61 Success in
treating infections in animal models of cystic fibrosis associated
112-9
infection was also reported by Debarbieux et al62 and Carmody
et al63 in addition to the exploration of using nebulization as a phage
delivery strategy. Phage treatments of lung infections, however, can
also be used effectively, in at least some circumstances from systemic
circulation, as animal models have shown.63
Ear Infections
Chronic otitis externa, known less formally as swimmer’s ear, is
often caused by a P. aeruginosa ear infection that resists antibiotic
treatment. Otitis media is often caused by Streptococcus pneumoniae,
and is the leading cause of physician visits from children. The company, Biocontrol (recently acquired by Targeted Genetics of Seattle
to form a new joint company, AmpliPhi Biosciences) has been
developing anti-P. aeruginosa phages targeting otitis externa, after
publishing similar studies on dogs.64,65 In 2009, they published the
results of their double-blinded phase 1/2a (safety and small-number
efficacy) trial in human patients with this condition.66 Increases in
phage numbers in situ, microbiological improvements (reductions
in bacterial presence), and reduction in disease symptoms in the
phage-treated cohort, but not the phage-negative controls, were
observed. No side effects were seen. Complete bacterial eradication
was not observed, but the extent of success was particularly notable
considering that only a single phage dose was administered. WeberDabrowska et al57 also reported phage therapy success in treated
purulent otitis media, and Slopek et al58 reported 93.8% positive
results for phage treatment of 16 cases of conjunctivitis, blepharoconjunctivitis, and otitis media.
TOXICOLOGY
From a clinical standpoint, all indications are that phages are very
safe. This feature is not surprising, given that humans are exposed
to phages from birth. Bergh et al67 reported that nonpolluted
water contains about 108 phages/mL. Phages are normally found
in the GI tract, skin, urine, and mouth, where they are harbored
in saliva and dental plaque.68-70 They have also been shown to be
unintentional contaminants of sera and therefore of commercially
available vaccines,71-74 which were given dispensation to be sold
despite this discovery, because of the general consensus that phages
are safe for humans.
Extensive preclinical animal testing was required for approving
new phage formulations in the former Soviet Union, but few of
these studies were published. For example, Bogovazova et al75,76
evaluated the safety and efficacy of Klebsiella phages produced by
the Russian company Immunopreparat. Pharmacokinetic and
toxicologic studies using intramuscular, intraperitoneal, or intravenous administration of phages were carried out in mice and
guinea pigs. The researchers found no signs of acute toxicity or
histologic changes, even using a dose 3500-fold higher than the
projected human dose. They then evaluated the safety and efficacy
of the phages in treating 109 patients. The phage preparation was
reported to be nontoxic to humans and to be effective in treating
Klebsiella infections, as manifested by marked clinical improvements and bacterial clearance in the phage-treated patients.
Occasional mild side effects such as liver pain and fever reported
in the early days of Western phage therapy may have been due to
bacterial by-products in preparations used intravenously.77-79
Concerned about this possibility, the Polish group does not
administer their phages intravenously. The same is true for almost
all of the therapeutic work carried out in Tbilisi, and perhaps
helps to explain the virtually total lack of significant problems in
both places; their many years of experience, careful attention to
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
112-10
SECTION 5
|
PHARMACOLOGY OF NATURAL MEDICINES
detail, and supportive infrastructure are presumably also important factors. Because phages readily enter the bloodstream after
infusion in or near wounds and other sites of localized infection
and travel to sites of infection throughout the body,80 there generally seems to be no reason for undergoing the extra risks of intravenous administration.
Drug Interactions
No negative effects on the efficacy or safety of other drugs have been
reported anywhere as a result of phage administration. No systematic studies have been carried out in this regard, but phages are so
specific in their actions that it is hard to determine where such interactions might occur. In contrast, at least some antibiotics can interfere with phage treatment of localized infections by killing off the
most accessible of the bacteria in which the phages need to multiply
as they work their way deeper into the lesion; this would be a particular problem in cases in which the phages can still attach and
infect but cannot complete their replication cycle. Georgian physicians generally believe that antibiotics should never be used topically
for wounds and deep-seated infections, because the decrease in antibiotic concentration below the surface provides a strong selection for
antibiotic resistance; this problem does not occur with phages.
DOSING STRATEGY
Phage cocktails can be designed in two distinctly different ways.
The major style currently used in Georgia, Russia, and Poland,
termed active treatment, uses a “low” concentration of phages so as
to rely on in situ phage replication to achieve a therapeutically relevant concentration of phage. Titers of individual phages are typically approximately 106 to 107 PFU/mL and in general 5 to 10 mL
is used per dose. This approach is preferred particularly when antibiotics fail due to poor circulation or surgical inaccessibility. The
other style, passive treatment, ignores the self-replicating nature of
phages in favor of a more conventional dosing strategy, in which
the infected area is directly accessible and a sufficient number of
particles are applied to treat the infection in a single dose.81
CONCLUSION
Phages have many potential advantages:
• They are self-replicating but also self-limiting, because they
multiply only in the presence of sensitive bacteria.
• They can be targeted much more specifically than most antibiotics to the problem bacteria, causing far less of the bacterial
imbalance or “dysbiosis” that are major problems with antibiotics, often leading to serious secondary infections involving relatively resistant bacteria that can increase hospitalization time,
expense, and mortality (see Chapters 10 and 27). Particular
resultant problems are Pseudomonads, which are especially difficult to treat, and Clostridium difficile, the cause of serious diarrhea and membranous colitis.82
• Phages can often be targeted to receptors on the bacterial surface that are involved in pathogenesis, so any resistant mutants
are less problematic.
• No serious side effects have been reported for phage therapy.
• Phage therapy could be particularly useful for people with allergies to antibiotics.
• Appropriately selected phages can easily be used prophylactically to help prevent bacterial disease at times of exposure or to
sanitize hospitals and help protect against hospital-acquired
(nosocomial) infections.
• Especially for external applications, phages can be prepared
fairly inexpensively and locally, facilitating their potential applications to underserved populations.
• Phages can be used either independently or in conjunction with
other antibiotics to help reduce the development of bacterial
resistance.
The time has come to look more carefully at the potential of
phage therapy for future practice, both by strongly supporting new
research and by scrutinizing the research already available, such as
the very interesting human antityphoid phage research carried out
in this country in the 1940s,30 as well as the extensive earlier work
in France, the United States,Georgia, Poland, and Russia.4,5
With the enormous possibilities and decreasing costs of genomic
analysis, it is now possible to perform genomic sequencing of the
phages included in cocktails to ascertain more about the phage
families involved and exclude phages from temperate families,
because they are likely to carry or acquire genes related to pathogenicity or toxin production. This is now standard procedure for
therapeutic phages being developed in the West. Such modern
techniques are beginning to be applied to some of the Georgian
phage preparations with help from grants from the International
Science and Technology Centers and Civilian Research and Development Foundation programs, both of which were set up to support civilian applications of science formerly funded by the Soviet
military. This is an important step in considering the importation
of such phages for topical use in the Western world.
Although it seems premature to broadly introduce injectable
phage preparations in the West without further extensive research,
their carefully implemented use in external applications and for a
variety of agricultural purposes could potentially help reduce the
emergence of antibiotic-resistant strains and deal with problems
we have difficulty handling today. Furthermore, compassionate use
of appropriate phages seems warranted in cases in which bacteria
resistant to all available antibiotics are causing life-threatening illness. Phages are especially useful in dealing with recalcitrant nosocomial infections, in which large numbers of particularly vulnerable
people are being exposed to the same strains of bacteria in a closed
hospital setting. In these cases especially, the environment as well as
the patients, can be effectively treated with phage preparations.
ACKNOWLEDGMENTS
Special thanks to Drs. Liana Gachechiladze, Amiran Meipariani,
Guram Gvasalia, Ramaz Katsarava, Mzia Kutateladze, Rezo Adamia,
Teona Danelia, Naomi Hoyle, and their colleagues in Tbilisi, and to
Beata Weber-Dabrowski and Andre Gorski in Wroclaw for their
hospitality, hard work on phage therapy, and efforts through the
years to help us understand the extensive therapeutic work carried
out there. We also express our thanks to the many phage biologists
and health care personnel now working to bring phage therapy back
into the Western World, particularly Harald Brüssow.
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
CHAPTER 112
|
Phage Therapy: Bacteriophages as Natural, Self-Limiting Antibiotics
112-11
REFERENCES
1. “World Health Day- 7 April 2011,
Bacterial resistance: no action today, no
cure tomorrow.” www.who.int/world-healthday/2011/en. Accessed 9/19/2011.
2. Häusler T. Viruses vs. Superbugs: A
Solution to the Antibiotic Crisis. New York:
Macmillan; 2006.
3. Kutter E. Bacteriophage Therapy: Past and
Present. In: Schaechter M, ed. Encyclopedia of Microbiology. New York: Elsevier;
2009:258-266.
4. Abedon S, Kuhl S, Blasdel B, et al. Phage
Treatment of Human Infections. Bacteriophage. 2011;1:66-85.
5. Kutter E, De Vos D, Gvasalia G, et al.
Phage therapy in clinical practice:
treatment of human infections. Curr
Pharm Biotechnol. 2010;11:69-86.
6. Sulakvelidze A, Kutter E. Bacteriophage
therapy in humans. In: Kutter E,
Sulakvelidze A, eds. Bacteriophages:
Biology and Application. Boca Raton,
Florida: CRC Press; 2005:381-436.
7. Brüssow H. Phage therapy: the Western
perspective. In: Mc Grath S, van Sinderen
D, eds. Bacteriophage: Genetics and
Microbiology. Norfolk, UK: Caister
Academic Press; 2007:159-192.
8. Górski A, Borysowski J, Miedzybrodzki R,
et al. Bacteriophages in medicine. In:
Mc Grath S, van Sinderen D, eds.
Bacteriophage: Genetics and Microbiology.
Norfolk, UK: Caister Academic Press;
2007:125-158.
9. Harper DR, Kutter E. Bacteriophage:
therapeutic use. Encyclopedia of Life
Sciences. Hoboken, NJ: John Wiley &
Sons, Ltd.; 2009:1-724.
10. Górski A, Miedzybrodzki R, Borysowski J,
et al. Bacteriophage therapy for the
treatment of infections. Curr Opin Investig
Drugs. 2009;10:766-774.
11. Alisky J, Iczkonski K, Rapoport A, et al.
Bacteriophage shows promise as antimicrobial agents. J Infection. 1998;36:5-13.
12. Sulakvelidze A, Alavidze A, Morris J.
Bacteriophage therapy. Antimicrob Agents
Chemother. 2001;45:649-659.
13. Summers WC. Felix d’Herelle and the
origins of molecular biology. New Haven,
CT: Yale University Press; 1999.
14. Kutter E, Sulakvelidze A. Bacteriophages:
biology and applications. Boca Raton, FL:
CRC Press; 2004.
15. Antibacterial treatment against diarrhea in
oral rehydration solution. http://www.
clinicaltrials.gov/ct2/show/NCT00937274.
Accessed 3/04/2012.
16. Chibani-Chennoufi S, Sidoti J, Bruttin A,
et al. Isolation of T4-like bacteriophages
from the stool of pediatric diarrhea
patients in Bangladesh. J Bacteriol.
2004;186:8287-8294.
17. Bruttin A, Brüssow H. Human volunteers
receiving Escherichia coli phage T4 orally:
a safety test of phage therapy. Antimicrob
Agents Chemother. 2005;49:2874-2878.
18. D’Herelle F (Smith GH, trans). The
bacteriophage: its role in immunity.
Baltimore: Williams & Wilkins; 1922.
19. R, Maisin J. Essais de therapeutique au
moyen du bacteriophage du staphylocoque.
C R Soc Biol. 1921;85:1120-1121.
20. Eliava G. Bakteriofagi fenomenvyz
dorovieniya. Tbilisi, Georgia: Tbilis
National University Publications; 1935.
21. Summers WC. Bacteriophage Therapy. Ann
Rev Microbiol. 2001;55:437-451.
21a. Straub ME, Appelbaum M. Studies on
commercial bacteriophage products.
JAMA. 1933;100:110-113.
22. Ruska H. Die sichtbarmachung der
bakteriophagen lyse im ubermikroskop.
Naturwissenschaften. 1940;28:45.
23. Pfankuch E, Kausche G, Isolierung U.
Uebermikroskopische abbildung eines
bakteriophagen. Naturwissenschaften.
1940;28:46.
24. Appelmans R. Le bacteriophage dans
l’organisme. C R Seances Soc Biol Fil.
1921;85:722-724.
25. Evans AC. Inactivation of antistreptococcus bacteriophage by animal fluids. Public
Health Reports 1933;48:411-46. Cited in
Merril C, S Adhya S. Phage therapy. In:
Calendar R, ed. The Bacteriophages.
2nd ed. Oxford: Oxford University Press;
2006:725-741, ipress.
26. Dubos RJ, Straus JH, Pierce C. The
multiplication of bacteriophage in vivo and
its protective effects against an experimental infection with Ishigella dysenteriae. J Exp Med. 1943;78:161-168.
27. Morton HE, Engely FB. Dysentery bacteriophage: review of the literature on its
prophylactic and therapeutic uses in man
and in experimental infections in animals.
JAMA. 1945;17:584-891.
28. Häusler T. Gesund durch Viren. München.
Germany: Piper; 2003.
29. Ward WE. Protective action of VI bacteriophage in Eberthella typhi infections in
mice. J Infect Dis. 1943;72:172-176.
30. Knouf EG, Ward WE, Reichle PA, et al.
Treatment of typhoid fever with type
specific bacteriophage. JAMA. 1946;132:
134-138.
30a. Desranleau JM. Progress in the treatment
of typhoid fever with Vi bacteriophages.
Can J Public Health. 1949 Nov;40(11):
473-478.
31. Vieu JF. Les bacteriphages. In: Fabre J, ed.
Traite de therapeutique, Serums et vaccins.
Paris: Flammarion; 1975:337-430.
32. Vieu JF, Guillermet F, Minck R, et al.
Données actuelles sure les applications
therapeutiques des bacteriophages. Bull
Acad Natl Med. 1979;163:61-66.
33. Slopek S, Durlakova I, Weber-Dabrowska B,
et al. Results of bacteriophage treatment of
suppurative bacterial infections. I. General
evaluation of the results. Arch Immunol
Ther Ezp. 1983;31:267-291.
34. Slopek S, Kucharewica-Krukowska A,
Weber-Dabrowska B, et al. Results of
bacteriophage treatment of suppurative
bacterial infections. VI. Analysis of
treatment of suppurative staphyulococcal
infections. Arch Immunol Ther Exp.
1985;33:261-273.
35. Slopek S, Weber-Dabrowska B, Dabrowski
M, et al. Results of bacteriophage
treatment of suppurative bacterial
infections in the years 1981-1986. Arch
Immunol Ther Exp. 1987;35:569-583.
36. Weber-Dabrowska B, Debrowska M, Slopek
S. Studies on bacteriophage penetration in
patients subjected to phage therapy. Rch
Immunol Ther Exp (Warsz). 1987;35:
363-368.
37. Smith HW, Huggins RB. Successful
treatment of experimental E. coli
infections in mice using phage: its general
superiority over antibiotics. J Gen
Microbiol. 1982;128:307-318.
38. Smith HW, Huggins RB. Effectiveness of
phages in treating experimenta E. coli
diarrhoea in calves, piglets and lambs.
J Gen Microbiol. 1983;129:2659-2675.
39. Smith HW, Huggins RB. The control of
experimental E. coli diarrhea in calves by
means of bactgeriophage. J Gen Microbiol.
1987;133:1111-1126.
40. Smith HW, Huggins RB, Shaw KM.
Factors influencing the survival and
multiplication of bacteriophages in calves
and in their environment. J Gen Microbiol.
1987;133:1127-1135.
41. Weber-Dabrowska B, Mulczyk M, Gorski A.
Bactgeriophage therapy of bacterial
infections: an update of our institute’s
experience. Arch Immunol Ther Exp.
2000;48:547-551.
42. Katsarava R, Beridze V, Arabuli N, et al.
Amino acid-based bioanalogous polymers.
Synthesis and study of regular poly (ester
amide)s based on bis(a-amino acid),
alpha, omega-alkylene diesters, and
aliphatic dicarboxylic acids. J Polymer Sci.
1999;37:391-407.
43. Markoishvili K, Tsitlanadze G, Katsarava R,
et al. A novel sustained-release matrix
based on biodegradable poly (ester amide)s
and impregnated with bacteriophages and
an antibiotic shows promise in management of infected venous stasis ulcers and
other poorly healing wounds. Int J
Dermatol. 2002;41:453-458.
44. MacNeal WJ, Frisbee FC. One hundred
patients with Staphylococcus septicemia
receiving bacterio-phage service. Am J
Med Sci. 1936;191:179-195.
45. Eaton MD, Bayne-Jones S. Bacteriophage
therapy: Review of the principles and
results of the use of bacteriophage in the
treatment of infections (I). JAMA.
1934;103:1769-1776.
To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and
typesetter TNQ Books and Journals Pvt Ltd. It is not allowed to publish this proof online or in print. This proof copy is the copyright property of the publisher and is confidential until formal
publication.
112-12
SECTION 5
|
PHARMACOLOGY OF NATURAL MEDICINES
46. Eaton MD, Bayne-Jones S. Bacteriophage
therapy: Review of the principles and
results of the use of bacteriophage in the
treatment of infections (II). JAMA.
1934;103:1847-1853.
47. Eaton MD, Bayne-Jones S. Bacteriophage
therapy: Review of the principles and
results of the use of bacteriophage in the
treatment of infections (III). JAMA.
1934;103:1934-1939.
48. MacNeal WJ, Frisbee FC, McRae MA.
Staphylococcemia 1931–1940. Five
Hundred Patients. Am J Clin Pathol.
1942;12:281-294.
49. Kutter E. Phage Therapy: Bacteriophages
as naturally occurring antimicrobials. In:
Goldman E, Green LH, eds. Practical
Handbook of Microbiology. Boca Raton,
FL: CRC Press; 2008:713-730.
50. Bruynoghe R, Maisin J. Essais de
thérapeutique au moyen du bactériophage
du Staphylocoque. Compt Rend Soc Biol.
1921;85:1120-1121.
51. Kutter E, Sulakvelidze A, eds. Bacteriophages: Biology and Application. Boca
Raton, FL: CRC Press; 2005:335-380.
51a. Nathan Brown, personal communication.
2009.
51b. Mann NH. The potential of phages to
prevent MRSA infections. Res Microbiol.
2008;159:400-405.
51c. O’Flaherty S, Ross RP, Meaney W, et al.
Potential of the polyvalent antiStaphylococcus bacteriophage K for
control of antibiotic-resistant staphylococci from hospitals. Appl Environ
Microbiol. 2005;71:1836-1842.
51d. Leszczynski P, Weber-Dabrowska B,
Kohutnicka M, et al. Successful eradication of methicillin-resistant Staphylococcus aureus (MRSA) intestinal carrier
status in a healthcare worker - a case
report. Folia Microbiol. 2008;
51:236-238.
52. Miedzybrodzki R, Fortuna W, Weber-Dabrowska B, et al. Phage therapy of
staphylococcal infections (including
MRSA) may be less expensive than
antibiotic treatment. Postepy higieny i
medycyny doswiadczalnej. 2007;
61:461-465.
53. Abdul-Hassan HS, El-Tahan K, Massoud B,
et al. Bacteriophage therapy of pseudomonas burn wound sepsis. Ann Med Burn Club.
1990;3:262-264.
53a. Jikia D, Chkhaidze N, Imedashvili E,
et al. The use of a novel biodegradable
preparation capable of the sustained
release of bacteriophages and ciprofloxacin, in the complex treatment of multidrug-resistant Staphylococcus
aureus-infected local radiation injuries
caused by exposure to Sr90. Clin Exp
Dermatol. 2005;30:23-26.
54. Letkiewicz S, Miedzybrodzki R, Fortuna W,
et al. Eradication of Enterococcus faecalis
by phage therapy in chronic bacterial
prostatitis—case report. Folia Microbiol
(Praha). 2009;54:457-461.
55. Letarov AV, Golomidova AK, Tarasyan KK.
Ecological basis of rational phage therapy.
Acta Naturae. 2010;2:60-71.
56. Chanishvili N. Phage therapy in urology.
In: Chanishvili N, Sharp R, eds. A
Literature Review of the Practical
Application of Bacteriophage Research.
Tbilisi: Georgia: Eliava Institute; 2009:
59-60.
56a. Alavidze. Personal correspondence.
2011.
57. Weber-Dabrowska B, Mulczyk M, Górski A.
Bacteriophage therapy for infections in
cancer patients. Clin Appl Immunol Rev.
2001;1:131-134.
58. Slopek S, Weber-Dabrowska B, Dabrowski
M, et al. Results of bacteriophage
treatment of suppurative bacterial
infections in the years 1981–1986. Arch
Immunol Ther Exp (Warsz). 1987;35:569583.
59. Kutateladze M, Adamia R. Bacteriophages
as potential new therapeutics to replace or
supplement antibiotics. Trends Biochem.
2010;28:591-595.
60. Kvachadze L, Balarjishvili N, Meskhi T,
et al. Evaluation of lytic activity of
staphylococcal bacteriophage Sb-1 against
freshly isolated clinical pathogens. Microb
Biotechnol. 2011:4-1-8.
61. Kutter E, De Vos D, Gvasalia G, et al.
Phage therapy in clinical practice:
treatment of human infections. Curr
Pharm Biotechnol. 2010;11:69-86.
62. Debarbieux L, Leduc D, Maura D, et al.
Bacteriophages can treat and prevent
Pseudomonas aeruginosa lung infections.
J Infect Dis. 2010;201:1096-1104.
63. Carmody LA, Gill JJ, Summer EJ, et al.
Efficacy of bacteriophage therapy in a
model of Burkholderia cenocepacia
pulmonary infection. J Infect Dis.
2010;201:264-271.
64. Marza JAS, Soothill JS, Boydell P, et al.
Multiplication of therapeutically administered bacteriophages in Pseudomonas
aeruginosa infected patients. Burns.
2006;32:644-646.
65. Hawkins C, Harper D, Burch D, et al.
Topical treatment of Pseudomonas
aeruginosa otitis of dogs with a bacteriophage mixture: A before/after clinical trial.
Vet Microbiol. 2010;146:309-313.
66. Wright A, Hawkins CH, Anggård EE, et al.
A controlled clinical trial of a therapeutic
bacteriophage preparation in chronic otitis
due to antibiotic-resistant Pseudomonas
aeruginosa; a preliminary report of
efficacy. Clin Otolaryngol. 2009;34:
349-357.
67. Bergh O, Borsheim KY, Bratbak G, et al.
High abundance of viruses found in
aquatic environments. Nature. 1989;340:
467-468.
68. Caldwell JA. Baceriologic and bacteriophagic study of infected urines. J Infect
Dis. 1928;43:353-362.
69. Yeung MK, Kozelsky CS. Transferction of
Actinomyces spp. By genomic DNA of bacteriophages from human dental plaque.
Plasmid. 1997;35:141-153.
70. Bachrach G, Leizerovici-Zigmond M,
Zlotkin A, et al. Bacteriophage isolation
from human saliva. Lett Appl Microbiol.
2003;36:50-53.
71. Merril CR, Friedman TB, Attallah AF, et al.
Isolation of bacterophages from commercial sera. In Vitro. 1972;8:91-93.
72. Geier MR, Attallah AF, Merril CR.
Characterization of escherichia coli
bacterial viruses in commercial sera. In
Vitro. 1975;11:55-58.
73. Milch H, Fornosi F. Bacteriophage
contamination in live poliovirus vaccine.
J Biol Stand. 1975;3:2307-2310.
74. Moody EE, Trousdale MD, Jorgensen JH,
et al. Bacteriophages and endotoxin in
licensed live-virus vaccines. J Infect Dis.
1975;131:588-591.
75. Bogovazova GG, Voroshilova NN, Bondarenko VM. The efficacy of Klebsiella
pneumoniae bacteriophage in the therapy
of experimental Klebsiella infection. Zh
Mikrobiol Epidemiol Immunobiol.
1991;4:5-8:Language: Russian.
76. Bogovazova GG, Voroshilova NN, Bondarenko VM, et al. Immunobiological
properties and therapeutic effectiveness of
preparations from Klebsiella bacteriophages. Zh Mikrobiol Epidemiol Immunobiol. 1992;3:30-33:Language: Russian.
77. Larkum NW. Bacteriophage as a substitute
for typhoid vaccine. J Bacteriol. 1929;
17:42.
78. Larkum NW. Bacteriophage from Public
Health standpoint. Am J Pub Health.
1929;19:31-36.
79. King WE, Boyd DA, Conlin JH. The cause
of local reactions following the administration of Staphylococcus bacteriophage. Am
J Clin Pathol. 1934;4:336-345.
80. Górski A, Ważna E, Weber-Dąbrowska B,
et al. Bacteriophage translocation. FEMS
Immunol Med Microbiol. 2006;46:
313-319.
81. Abedon ST, Thomas-Abedon C. Phage
therapy pharmacology. Curr Pharm
Biotechnol. 2010;11:28-47.
82. Fekety R. Antibiotic-associated diarrhea
and colitis. Cur Opin Infect Dis. 1995;
8:391-397.
Our reference: Chapter 112
P-authorquery-v3
AUTHOR QUERY FORM
Chapter: 112
Please e-mail or fax your responses and any corrections to:
Dear Author,
During the preparation of your manuscript for typesetting, some questions may have arisen. These are listed below. Please check
your typeset proof carefully and mark any corrections in the margin of the proof or compile them as a separate list*.
Disk use
Sometimes we are unable to process the electronic file of your article and/or artwork. If this is the case, we have proceeded by:
Scanning (parts of) your article
Rekeying (parts of) your article
Scanning the artwork
Bibliography
If discrepancies were noted between the literature list and the text references, the following may apply:
The references listed below were noted in the text but appear to be missing from your literature list. Please complete the
list or remove the references from the text.
Uncited references: This section comprises references that occur in the reference list but not in the body of the text. Please
position each reference in the text or delete it. Any reference not dealt with will be retained in this section.
Queries and/or remarks
Location in article
Query / remark
No Queries
Thank you for your
assistance
Response