Phage Therapy
Phage as Antibiotics
PHAGE THERAPY: BACTERIOPHAGES AS ANTIBIOTICS
Elizabeth
Kutter, Evergreen State College,
t4phage@evergreen.edu; 360 867-6099
or 867-6523
Bacteria resistant to most or all available antibiotics are causing increasingly serious problems, raising widespread fears of returning to a pre-antibiotic era of untreatable infections and epidemics. Despite intensive work by drug companies, no new classes of antibiotics have been found in the last 30 years. There are hopes that the newfound ability to sequence entire microbial genomes and to determine the molecular bases of pathogenicity will open new avenues for treating infectious disease, but other approaches are also being sought with increasing fervor. One result is a renewed interest in the possibilities of bacteriophage therapy -- the harnessing of a specific kind of viruses that attack only bacteria to kill pathogenic microorganisms (cf. Levin and Bull, 1996; Lederberg, 1996; Radetsky, 1996; Barrow and Soothill, 1997).
Phage therapy
was first developed early in this century and showed much promise but also
aroused much controversy. It has been little used in the West since the advent
of antibiotics in the 1940s. However, extensive clinical research and
implementation of phage therapy continued to be carried out in
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Viruses are like space ships that are able to carry genetic material between susceptible cells and then reproduce in those cells, just as the AIDS virus, HIV, specifically infects human T lymphocytes which carry a particular surface protein called CD4. Each virus consists of a piece of genetic information, determining all of the properties of the virus, which is carried around packaged in a protein coat. In the case of bacteriophages, the targets are specific kinds of bacterial cells; they cannot infect the cells of more complex organisms because of major differences in key intracellular machinery as well as in cell-surface proteins. Most phages have tails, the tips of which have the ability to bind to specific molecules on the surface of their target bacteria. The viral DNA is then injected through the tail into the host cell, where it directs the production of progeny phages -- often over a hundred in half an hour. Each strain of bacteria has characteristic protein, carbohydrate and lipopolysaccharide molecules present in large quantities on its surface. These molecules are involved in forming pores, in motility, in 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 loss of its specific receptor; this loss often has negative effects on the bacterium and does not protect it against the many other phage which use different receptors.
Each
kind of bacteria has its own phages, which can be isolated wherever that
particular bacterium grows -- from sewage, feces, soil, even ocean depths and
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Discovery
A
century ago, Hankin (1896)
reported that the waters of the Ganges and Jumna rivers in
D'Herelle
went on to carefully characterize bacteriophages as viruses which multiply in
bacteria and worked out the details of infection by various phages of different
bacterial hosts under a variety of environmental conditions, always working to
combine natural phenomena with laboratory findings, to better understand
immunity and natural healing from infectious disease (Summers,
1998). The Ninetieth Annual Meeting of the British Medical Association in
D'Herelle summarized the early phage work in a 300-page book "The Bacteriophage" (1922). He wrote 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 sources of infectious bacteria 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. He noted for example the frequent specificities of the phages isolated from recuperating patients for their own disease organisms and the rather rapid variations over time in their phage populations. He thus worked throughout his life to develop the potential of using properly selected phages as therapeutic agents against the most devastating health problems of the day. However, he initially focussed on simply understanding phage biology. Thus, the first known report of successful phage therapy came not from d'Herelle but from Bruynoghe and Maisin (1921), who used phage to treat staphylococcal skin infections.
After
a year at the Pasteur Institute of Saigon, d'Herelle returned to tight physical
conditions, personal conflict and intellectual controversy at the Pasteur
Institute in
In
1928, d'Herelle was invited to Stanford to give the prestigious Lane Lectures;
his discussion of "The Bacteriophage and its Clinical Applications"
was published as a monograph (d'Herelle and Smith, 1930). He gave many lectures
for medical schools and societies as he crisscrossed the country. He then went
on to Yale to take up a regular faculty position, arranged with the support of
George Smith, who had translated his first two books into English. He continued
to spend summers in
From early on, one major practical use of phages was for bacterial identification through a process called phage typing -- the use of patterns of sensitivity to a specific battery of phages to precisely identify microbial strains. This technique takes advantage of the fine specificity of many phages for their hosts and is still in common use around the world. The sophisticated ability of phages to destroy their bacterial hosts can also have a very negative commercial impact; phage contaminants occasionally spread havoc and financial disaster for the various fermentation industries that depend on bacteria, such as cheese production and fermentative synthesis of chemicals (cf. Saunders, 1994)
Phage therapy was tried extensively and many successes were reported for a variety of diseases, including dysentery, typhoid and paratyphoid fevers, cholera, and pyogenic (pus-producing) and urinary-tract infections. Phages were poured directly into lesions, given orally or applied as aerosols or enemas. They were also given as injections -- intradermal, intravascular, intramuscular, intraduodenal, intraperitoneal, even into the lung, carotid artery and pericardium. The early strong interest in phage therapy is reflected in the fact that some 800 papers were published on the topic between 1917 and 1956; the results have been discussed in some detail by Ackermann and Dubow (1987). The reported results were quite variable. Many physicians and entrepreneurs became very excited by the potential clinical implications and jumped into applications with very little understanding of phages, microbiology or 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 much scientific sense. Often, uncharacterized phages at unknown concentrations were given to patients without specific bacteriological 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 (Summers,
1998). On another occasion, a preparation was advertised as containing a
number of different phages, but it turned out that the technician responsible
had decided it was easier to grow them up in one large batch than in separate
batches. Not too surprisingly, checking the product showed that one phage had
outcompeted all the others and this was not, in fact, a polyvalent preparation.
This was the origin of the phage T7, whose RNA polymerase now plays a major
role in biotechnology (William Summers, personal communication). In general,
there was no quality control except in a few research centers. Large clinical
studies were rare and the results of those few were largely inaccessible
outside of
In 1931, an extensive review of bacteriophage therapy was commissioned by the Council on Pharmacy and Chemistry of the American Medical Association (Eaton and Bayne-Jones, 1931). Its purpose was "(a) to present summaries and discussions of (1) the experimentally determined facts relating to the bacteriophage phenomenon, (2) the laboratory and clinical evidence for and against the therapeutic usefulness of bacteriophage and (3) the relation of so-called antivirus to materials containing bacteriophage, and (b) to serve as a basis for a survey of the status of some of the commercial preparations." With 150 references, this report made a major effort to survey at least what they considered the most significant papers and reviews. In evaluating this report, it is important to realize how little was yet known then about bacteriophages. In fact, their first conclusion was "Experimental studies of the lytic agent called "bacteriophage" have not disclosed its nature. D'Herelle's theory that the material is a living virus parasite of bacteria has not been proved. On the contrary, the facts appear to indicate that the material is inanimate, possibly an enzyme." In retrospect, the proof that phages are viruses looks solid and it is hard to see how they could have come to this conclusion, which clearly impacted all of their other findings. These included: "2.) Since it has not been shown conclusively that bacteriophage is a living organism, it is unwarranted to attribute its effect on cultures of bacteria or its possible therapeutic action to a vital property of the substance. 3.) While bacteriophage dissolves sensitive bacteria in culture and causes numerous modifications of the organisms, its lytic action in the body is inhibited or greatly impeded by blood and other bodily fluids. 4.) The material called bacterophage is usually a filtrate of dissolved organisms, containing, in addition to the lytic principle, antigenic bacterial substances, products of bacterial growth and constituents of the culture medium. The effects of all these constituents must be taken into consideration whenever therapeutic action is tested. 5) A review of the literature on the use of bacteriophage in the treatment of infections reveals that the evidence for the therapeutic value of lytic filtrates is for the most part contradictory. Only in the treatment of local staphylococcic infections and perhaps cystitis has evidence at all convincing been presented."
This
assessment clearly had a strong influence on the investment of the medical
community in exploring phage therapy seriously, at least in the
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SPECIFIC PROBLEMS OF EARLY PHAGE THERAPY WORK
Today, many believe that phage therapy was proven not to work in the early part of this century. However, it appears that it simply was never given sufficient and appropriate trial, and reassessment is warranted. It is thus important to consider in some detail potential reasons for the early problems and the questions as to efficacy. These included:
- Paucity of understanding of the heterogeneity and ecology of both the phages and the bacteria involved.
- Failure to select phages of high virulence against the target bacteria before using them in patients.
- Use of single phages in infections which involved mixtures of different bacteria.
- Emergence of resistant bacterial strains. This can occur by selection of resistant mutants (a frequent occurrence if only one phage strain is used against a particular bacterium) or by lysogenization (if temperate phages are used, as discussed below).
- Failure to appropriately characterize or titer phage preparations, some of which were totally inactive.
- Failure to neutralize gastric pH prior to oral phage administration.
- Inactivation of phages by both specific and nonspecific factors in body fluids.
- Liberation of endotoxins as a consequence of widespread lysis of bacteria within the body (which physicians call the Herxheimer reaction). This can lead to toxic shock, and is a potential problem with chemical antibiotics as well.
- Lack of availability or reliability of bacterial laboratories for carefully identifying the pathogens involved, necessitated by the relative specificity of phage therapy.
In making the choice to again explore the possibilities of phage therapy, we should also consider their many potential advantages, discussed in more detail below:
- They are both self-replicating and self-limiting, since they will multiply only as long as sensitive bacteria are present and then are gradually eliminated from the individual and the environment.
- They can be targeted far more specifically than can most antibiotics to the specific problem bacteria, causing much less damage to the normal microbial balance in the body. The bacterial imbalance or "dysbiosis" caused by treatment with many antibiotics can lead to serious secondary infections involving relatively resistant bacteria, often extending hospitalization time, expense and mortality. Particular resultant problems include infection by pseudomonads, which are especially difficult to treat, and Clostridium difficile, cause of serious diarrhea and membranous colitis (cf. Fékéty, 1995).
- Phages can often be targeted to receptors on the bacterial surface which are involved in pathogenesis, so that any resistant mutants are attenuated in virulence.
- Few side effects have been reported for phage therapy.
- Phage therapy would be particularly useful for people with allergies to antibiotics.
- Appropriately selected phages can easily be used prophylactically to help prevent bacterial disease in people or animals 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.
- Phage can be used either independently or in conjunction with other antibiotics to help reduce the development of bacterial resistance.
One major source of confusion in the early phage work was the perception that all phages were fundamentally similar, though subject to adaptive change depending on the recent conditions of growth. One consequence of this was that often new phages were isolated for each series of studies, so that there was little continuity or basis for comparison. Phages specific for over 100 bacterial genera have now been isolated (Ackermann, 1996); they have been found virtually everywhere that they have been sought. However, only few have yet been well studied or classified (cf. Ackermann and DuBow, 1987)
A second early source of confusion affecting therapeutic uses was the question of whether the lytic principle termed "bacteriophage" simply reflected an inherent property of the specific bacteria or required regular reinfection by an external agent. During the 1930s and 1940s, it became increasingly clear that in some senses both were true -- that there were in fact two quite fundamentally different groups of bacteriophages. 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 interval. Lysogenic phages, on the other hand, have another option. They can actually integrate their DNA into the host DNA, much as HIV can integrate the DNA copy of its RNA, leading to virtually permanent association as a prophage with a specific bacterium and all its progeny. The prophage directs the synthesis of a repressor, which blocks the reading of the rest of its own genes and also those of any closely-related lysogenic phages -- a major advantage for the bacterial cell. Many prophages further aid their host by helping protect against various unrelated, lytic phages. Occasionally, a prophage escapes from regulation by the repressor, cuts its DNA back out of the genome by a sort of site-specific recombination and goes ahead to make progeny phage and lyse open the cell. Sometimes the cutting-out process makes mistakes and a few bacterial genes get carried along with the phage DNA to its new host; this process, called transduction, plays a significant role in bacterial genetic exchange. Such lysogenic phages are very bad candidates for phage therapy, both due to their mode of inducing resistance and to the fact that they can potentially lead to transfer of genes involved in bacterial pathogenicity; this is discussed in more detail below. However, their specificity often makes them very useful for phage typing in distinguishing between bacterial strains.
Key technical developments that
helped clarify the general nature and properties of bacteriophages included:
(1) the concentration and purification of some large phages by means of
high-speed centrifugation and the demonstration that they contained equal
amounts of DNA and protein (Schlesinger,
1933 a, b) and (2) visualization of phages by means of the electron
microscopic (EM) (Ruska,1940;
Pfankuch
and Kausche, 1940). Soon after, Ruska (1943)
reported the first attempts to use the EM for phage systematics; this has since
become a key tool of the field (cf. Ackermann
and DuBow, 1987). Each phage was found to have its own specific shape and
size, from the "lunar lander"-style complexity of T4 and its
relatives to the globular heads with long or short tails of lambda and T7 to
the small filamentous phages that looked much like bacterial pili (See adjacent
figure, from Ackermann,
1996)
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A much better understanding of the interactions between lytic phage and bacteria came from detailed one-step growth curve experiments expanding on the work of d'Herelle (1922) (Ellis and Delbrück, 1939, Doermann,1952). These demonstrated an eclipse period during which the DNA began replicating and there were no free phage in the cell, a period of accumulation of intracellular phage, and a lysis process which released the phage to go in search of new hosts. An example of this phage infection cycle is outlined in the adjacent figure.
In
the early 1940's, developments occurred which were to have 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 Delbrück began working with key
phage biologists Alfred Hershey and Salvador Luria and formed the "Phage
Group", which eventually expanded dramatically with aid of the summer
Phage Courses at
The
influence of the Phage Group on the origins of molecular biology has been well
documented (cf. Cairns
et al, 1966; Fischer
and Lipson, 1988; Summers,
1993b). Virulent phages had just the right balance of complexity and
simplicity to tease out the key concepts of cell regulation at the molecular
level. However, a major element of the rapid success of phage as model systems
was that Delbrück convinced most phage biologists in the United States to focus
on one bacterial host (E. coli B) and 7 of its lytic phages, building a
very strong, tightly focussed community all working on the same set of
problems, able to build effectively on each other's work and communicate
easily. The 7 phages were arbitrarily chosen and named T(type)1-T7. As it
turned out, T2, T4 and T6 were quite similar to each other, defining the
"T-even" family of phages, discussed in more detail below. These
phages were key in demonstrating that DNA is the genetic material, that viruses
can encode enzymes, that gene expression is mediated through special copies in
the form of "messenger RNA", that the genetic code is triplet in
nature, and many other fundamental concepts. The negative side of this strong
focus on a few phages growing under rich laboratory conditions, however, was
that there was very little study or awareness of the ranges, roles and
properties of bacteriophages in the natural environment, or of phages that
infect other kinds of bacteria.
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The
rapid, powerful developments in the understanding of phage biology had 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 phage as tools to
understand molecular biology and those still working on phage ecology and
therapeutic applications. Many in the latter group were spurred on by concern
about the increasing incidence of nosocomial (hospital-acquired) infections and
of bacteria resistant against most or all known antibiotics. This is
particularly true in
Polish
Academy of Sciences ,
Wroclaw
The
most detailed publications documenting phage therapy have come from Stefan
Slopek's group at the
The
bacteriophages used all came from the extensive collection of the Bacteriophage
Laboratory of the
Few side effects were observed; those that were seen seemed directly associated with the therapeutic process. Pain in the liver area was often reported around day 3-5, lasting several hours; the authors suggested that this might be related to extensive liberation of endotoxins as the phage were destroying the bacteria most effectively. In severe cases with sepsis, patients often ran a fever for 24 hours about days 7-8 (Slopek et al, 1981a). Various other methods of administration were successfully used, including aerosols and infusion rectally or in surgical wounds. Intravenous administration was not recommended for fear of possible toxic shock from bacterial debris in the lysates (Slopek et al, 1981a). However, it was clear that the phages readily got into 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 (Weber-Dabrowska et al, 1987). This interesting and rather unexpected finding has been replicated in other studies and systems (** add refs.).
Detailed
notes were kept throughout on each patient. The final evaluating therapist also
filled out a special inquiry form that was sent to the
Bacteriophage
Institute,
The
most extensive and least widely known work on phage therapy was carried out
under the auspices of the Bacteriophage Institute at
According
to various Georgian physicians with whom I have spoken, phage therapy is part
of the general standard of care there, used especially extensively in
pediatric, burn and surgical hospital settings. Phage preparation was carried
out on an industrial scale, employing 1,200 people just before the break-up of
the
The
historical background of the institute is interesting, and reflects a
relatively unknown period in d'Herelle's caeer. The following material comes
from a number of people at the Institute, from a recent article by Shrayer (1996)
on d'Herelle in
In
1917, George Eliava, of the Georgian Institute of Microbiology, noticed that
the water of the Koura (Mtkvary) river in
In
1938, the Bacteriophage Institute was merged with the Institute of Microbiology
& Epidemiology, under direction of the People's Commissary of Health of
Georgia. In 1951, it was formally transferred to the All-Union Ministry of
Health set of Institutes of Vaccines and Sera, taking on the leadership role in
providing bacteriophages for therapy and bacterial typing throughout the former
This careful study of the host range, lytic spectrum and cross-resistance properties of the phages being used were a major factor in the reported successes of the phage therapy work carried out through the Institute. All of the phages used for therapy are lytic, avoiding the problems engendered by lysogeny. The problems of bacterial resistance were largely solved by the use of well-chosen mixtures of phages with different receptor specificities against each type of bacterium as well as of phages against the various bacteria likely to be causing the problem in multiple infections. The situation was further improved whenever the clinicians typed the pathogenic bacteria and monitored their phage sensitivity; where necessary, new cocktails were then prepared to which the given bacteria were sensitive. Not infrequently, using phage in conjunction with other antibiotics was shown to give better results than either the phage or the antibiotic alone.
The
depth and extent of the work involved is very impressive. For example, 1n
1983-85 alone, the Institute's Laboratory of Morphology and Biology of
Bacteriophages carried out studies of growth, biochemical features and phage
sensitivity of 2038 strains of Staphylococcus, 1128 of Streptococcus, 328 of
Proteus, 373 of Ps. aeruginosa and 622 of Clostridium, received from clinics
and hospitals in towns across the former
A
good deal of work has gone into developing and providing the documentation to
get approval from the Ministry of Health for 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 such as operating rooms. 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
Much
of the focus in the last 12 years has been on combating nosocomial infections,
where multi-drug-resistant organisms have become a particularly lethal problem
and where it is also easier to carry out proper long-term research. Clinical
studies of the effectiveness of the phage treatment and appropriate protocols
were carried out in collaboration with a number of hospitals, but little has
been published in accessible form. Zemphira Alavidze and her colleagues who are
currently doing most of the actual therapeutic development and clinical
application have manuscripts in preparation which describe their work in
institutions such as the Leningrad (St. Petersburg) Intensive Burn Therapy
Center, the Academy of Military Medicine in Leningrad, the Kazan Trauma Center,
the Kemerovo Maternity Hospital. Some of the most intensive studies were
carried out in
The Industrial Branch on the grounds of the Bacteriophage Institute had large vats for growing the selected phage, using appropriate nonpathogenic bacteria and broth they prepared themselves from high-quality beef. The resulting phage lysates were sterile filtered using ceramic filters which could themselves be sterilized in very hot ovens. The various different phages for each particular formulation were then combined and automatically packaged and sealed into 10-ml ampoules or otherwise prepared and packaged for administration. Approximate titers were determined by checking the dilution that would produce lysis after coinnoculation with specific numbers of bacteria of standard test strains, and each batch was also tested for any surviving bacterial contaminants. In those rare cases where injection was planned, the phages were concentrated and resuspended in physiological saline solution; testing in guinea pigs was added to the rest of the analytical regime, to make sure there were no residual bacterial surface fragments (endotoxins) that might cause problems if injected. (As mentioned above, phages have generally been reported to appear in the bloodstream and other body fluids rather shortly after being ingested or poured into a wound and to still be effective against systemic infections, so injection is usually not necessary.)
Injectable
forms accounted for only about 5% of the phage production at its height at the
Bacteriophage Institute. None are made there now due to such factors as the
expense and complexities of keeping animals for the necessary toxicity controls
in the difficult situation in
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RECENT WORK IN THE WEST RELATED TO PHAGE THERAPY
Levin and Bull (1996) and Barrow and Soothill (1997) have provided good reviews of much of the work applying phage therapy in animals which has been carried out in Britain and the United States since interest in the possibilities of phage therapy began to resurface there in the early 80's. The results in general are in very good agreement with the clinical work described above in terms of efficacy, safety and importance of appropriate attention to the biology of the host-phage interactions, reinforcing trust in the reported extensive eastern European results.
In Britain, H. W. Smith and M. B. Huggins (1982, 1983) carried out a series of studies on use of phages in systemic E. coli infections in mice and then in diarrheal disease in young calves. For example, they found that injecting 106 colony-forming units of a particular pathogenic strain intramuscularly killed 10/10 of the mice, but none died if they simultaneously injected 104 plaque-forming units of a phage selected against the K1 capsule antigen of that bacterial strain.This phage treatment was more effective than using such antibiotics as tetracycline, streptomycin, ampicillin or trimethoprim/sulfafurazole. Furthermore, the resistant bacteria that emerged had lost their capsule and were far less virulent. In calves, they found very high levels of protection even though they did not succeed in isolating phages specific for the K88 or K99 adhesive fimbriae, which play key roles in attachment within the small intestine. Still, the phage were able to reduce the number of bacteria bound there by many orders of magnitude and to virtually stop the fluid loss. The results were particularly effective if the phage were present before or at the time of bacterial presentation, and if multiple phages with different attachment specificities were used. Furthermore, the phage could be transferred from animal to animal, supporting the possibility of prophylactic use in a herd. If phage were given only after the development of diarrhea, the severity of the infection was still substantially reduced and none of the animals died (Smith et al, 1987). Levin and Bull (1996) carried out a detailed analysis of the population dynamics and tissue phage distribution of the 1982 Smith and Huggins study which can be helpful in assessing the parameters involved in successful phage therapy and its apparent superiority to antibiotics. They have gone on to do very interesting animal studies of their own (Levin and Bull, manuscript in preparation) and conclude that phage therapy is at least well worth further study.
Soothill (1994) carried out a series of very nice studies preparatory to using phages for infections of burn patients. Using guinea pigs, he showed that skin-graft rejection could be prevented by prior treatment with phage against Pseudomonas aeruginosa. He also saw excellent protection of mice against systemic infections with both Pseudomonas and Acinetobacter when appropriate phages were used (Soothill, 1992). In the latter case, as few as 100 phages protected against infection with 100 million bacteria -- 5 times the LD50!
Merrill
and coworkers (1996)
have carried out a series of experiments designed to better understand the
interactions of phages with the human immune system, and have started a company
called "Exponential Biotherapies, Inc." to explore the possibilities
of phage therapy. Their published work has been with a lytic derivative of the
lysogenic phage lambda. While this particular strain would be a poor choice for
therapeutic use, as discussed above and below, they have gathered very
interesting and important data about factors affecting interactions between
phages and the immune system.
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Most bacteria are not pathogenic; in fact, they play crucial roles in the ecological balance in various parts of our bodies, including the digestive system and all body surfaces. They actually help protect us from pathogens; this is one reason why the use of broad-spectrum antibiotics leaves us so vulnerable, and why more narrowly-targeted bactericidal agents would be highly advantageous. Furthermore, most of the serious pathogens are close relatives of non-pathogenic strains -- so what are the differences that make particular strains so lethal? Studies clarifying the mechanisms of pathogenesis at the molecular level have progressed remarkably in recent years (cf. Falkow 1996). These have now been crowned by the determination of the complete DNA base sequence of (nonpathogenic) E. coli K12 and several other bacterial species and extensive cloning and sequencing of pathogenicity determinants. Generally, a number of genes are involved, and these are clustered in so-called "pathogenicity islands", or "Pais", which may be 50,000-200,000 base pairs long. They generally have some unique properties indicating that the bacterium itself probably acquired them as a sort of "infectious disease" at some time in the past, and then kept them because they helped the bacterium infect new ecological niches where there was less competition. Many of these Pais are carried on small extrachromosomal circles of DNA called plasmids, which also can be carriers of drug-resistance genes. Others reside in the chromosome; there, they often are found imbedded in defective lysogenic prophages which have lost some key genes in the process and cannot be induced to form phage particles. However, they sometimes can recombine with related infecting phages. Therefore, it makes sense to avoid using lysogenic phages or their lytic derivatives for phage therapy to avoid any chance of picking up and moving such pathogenicity islands.
For bacteria in the human gut, pathogenicity involves 2 main factors: (1) the production of toxin molecules, such as shiga toxin (from Shigella and some pathogenic E. coli) or cholera toxin. These toxins modify proteins in the target host cells and thereby cause the problems. (2) the acquisition of new cell-surface adhesins which allow the bacterium to bind to specific receptor sites in the small intestine, rather than just moving on through to the colon. They also all contain the components of so-called type-III secretion machinery, related to those involved in assembly of flagella (for motility) and of filamentous phages and instrumental in many plant pathogens. For all of the pathogenic enteric bacteria, the infection process triggers changes in the neighboring intestinal cells. These include degeneration of the microvilli, formation of individual "pedestals" cupping each bacterium and, in the case of Salmonella and Shigella, induction of cell-signaling molecules that trigger engulfment of the bacterium and its subsequent growth inside the cell.
Recently,
E. coli O157 has been the subject of much concern, with contamination of such
products as hamburgers and unpasteurized fruit juices leading to serious
epidemics (cf. Grimm
et al., 1995). Deaths have occurred, particularly in young children and the
elderly, usually from hemorrhagic colitis (bloody diarrhea) or hemolytic-uremic
syndrome, where the kidneys are affected. Antibiotic therapy has shown no
benefit (cf. Greenwald
and Brandt, 1997). We find that the version of O157 from the
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A
substantial fraction of the phages in therapeutic mixes are relatives of
bacteriophage T4, which has played such a key role in the development of
molecular biology (cf. Karam, 1994). As discussed above, the name "T-even
family of phages" is a historical accident reflecting the fact that T2, T4
and T6 out of the original collection of Delbrück's "Phage Group" all
turned out to be related. Large sets of T4-like phages have been isolated for study
from all over the world -- for example, from Long Island sewage treatment
plants, animals in the
There have still been far too few studies of T4 ecology and its behavior under conditions more closely approaching the natural environment and the circumstances it will encounter in phage therapy, where the environment is often anaerobic and/or the bacteria experience frequent periods of starvation. The limited available information in that regard was summarized by Kutter, Kellenberger et al (1994). A variety of studies are shedding light on the ability of these highly virulent phages to coexist in balance with their hosts in nature. For example, they can reproduce in the absence of oxygen as long as their bacterial host had been growing anaerobically for several generations. We have found that they can also survive for a period of time in a sort of state of hibernation inside of starved cells and then allow their host to readapt enough when nutrients are again supplied to go on and produce a few phage. This is particularly interesting and important since bacteria undergo many drastic changes to survive periods of starvation which increase their resistance to a variety of environmental insults (cf. Kolter, 1992).
The T-even bacteriophages share a unique ability that contributes significantly to their widespread occurrence in nature and to their competitive advantage. They are able to control the timing of lysis in response to the relative availability of bacterial hosts in their environment. When E. coli cells are singly infected with T4, they lyse after 25-30 minutes at body temperature in rich media, releasing about 100-200 phage per cell. However, when additional T-even phages attack the cell more than 4 minutes after the initial infection, the cell does not lyse at the normal time. Instead, it continues to make phage for as long as 6 hours, with the exact time of eventual lysis affected by the multiplicity of superinfecting phage (cf. Doermann, 1948; Abedon, 1994). This delay is termed "lysis inhibition".
Thus,
for many reasons the T4-related family of phages make excellent candidates for
therapeutic use in enteric and other gram-negative bacteria, and studies of
their ecology and distribution are being carried out with these goals in mind
both in
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It is clearly time to look more carefully at the potential of phage therapy, both through strongly supporting new research and examining carefully what is already available. As Barrow and Soothill conclude, "Phage therapy can be very effective in certain conditions and has some unique advantages over antibiotics. With the increasing incidence of antibiotic resistant bacteria and a deficit in the development of new classes of antibiotics to counteract them, there is a need to investigate the use of phage in a range of infections." The stipulations of Ackermann (1987) are important here: "Blind treatment is clearly of no value; phages have to be tested just as antibiotics, and the indications have to be right, but this holds everywhere in medicine. However, phage therapy requires the creation of phage banks and a close collaboration between the clinician and the laboratory. Phages have at least one advantage....While the concentration of antibiotics decreases from the moment of application, phage numbers should increase. Another advantage is that phages are able to spread and thus prevent disease. Nonetheless, much research remains to be done ... on the stability of therapeutic preparations; clearance of phages from blood and tissues; their multiplication in the human body; inactivation by antibodies, serum or pus; and the release of bacterial endotoxins by lysis... In addition, therapeutic phages should be characterized at least by electron microscopy." While it seems premature to generally introduce injectible phage preparations in the West without further extensive research, their carefully-implemented use for a variety of agricultural purposes and in external applications could potentially help reduce the emergence of antibiotic-resistant strains. Furthermore, compassionate use of appropriate phages seems warranted in cases where bacteria resistant against all available antibiotics are causing life-threatening illness. They are especially useful in dealing with recalcitrant nosocomial infections, where large numbers of particularly vulnerable people are being exposed to the same strains of bacteria in a closed hospital setting. In this case, the environment as well as the patients can be effectively treated.
In 1925, Sinclair Lewis's classic novel Arrowsmith, for which he won the Nobel prize in literature, played a significant role in raising popular interest in the possibilities of phage therapy and the potential scientific and ethical dilemmas involved (Summers, 1991). Today, the growing scientific, public and commercial interest in phage therapy is being reflected and fanned in a number of ways. For example, the BBC recently produced a Horizon documentary on phage therapy, The Virus that Cures, building on the ideas in Radetsky's Discover article on Return of the Good Virus. Several companies are beginning to explore work with phage therapy. In addition, a nonprofit "PhageBiotics" foundation has been formed to help support communication, education and research in the field. Hopefully all of this attention will lead to increased support of badly-needed research in the field and to rapid progress in developing appropriate applications, providing at least one alternative to the growing problem of multi-drug-resistant bacteria.
Acknowledgments:
Special thanks to Drs. Rezo Adamia, Zemphira Alavidze, Teimuraz and Nino
Chanishvili, Taras Gabisonia, Liana Gachechiladze, Mzia Kutateladze, Amiran
Meipariani and their colleagues at the Bacteriophage Institute, Tbilisi, for
their hospitality and efforts to help me understand the extensive therapeutic
work carried out there. Others who have been particularly helpful with
information and communication include Dr. Marina Shubladze, pediatrician in
Tbilisi for 10 years, now residing in Seattle; Nino Mzavia, Nino Trapaidze,
Timur and Natasha Zurabishvili, who have worked in my laboratory on basic T4
biology; Hans-Wolfgang Ackermann (Laval University), Eduard Kellenberger
(Basel), William Summers (Yale), Steve Abedon (Ohio State) and Bruce Levin
(Emory); Mansour Samadpour, University of Washington; Kathy d'Acci, clinical
lab director, St. Peter's Hospital, Olympia); physicians Jess Spielholz, MD,
and Robin Moore, ND; and, especially, the many colleagues and students involved
in our laboratory at Evergreen, particularly Barbara Anderson, Pia Lippincott,
Mark Mueller, Stacy Smith, Elizabeth and Chelsea Thomas, Burt Guttman and Jim
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