Archivum
Immunologiae et Therapiae Experimentalis, 1999, 47, 267–274
PL ISSN 0004-069X
Review
Phage Therapy: Past History and Future
Prospects
RICHARD M. CARLTON
Exponential Biotherapies, Inc., 150 Main Street, Port Washington, NY 11050, USA
Abstract. Bacterial viruses (bacteriophages,
also called “phages”) can be robust antibacterial agents in vitro. However,
their use as therapeutic agents, during a number of trials from the1920s to the
1950s, was greatly handicapped by a number of factors. In part, there were
certain limitations inherent in phage physiology (e. g. narrow host range, and
rapid clearance from the body); in part there were technological limitations in
the era (e.g. lysogeny not yet discovered); but the greatest limitation was the
highly inadequate scientific methodologies used by practitioners at the time
(e.g., their failure to conduct placebo-controlled studies, to remove
endotoxins from the preparations, and to re-confirm phage viability after
adding sterilizing agents to the preparations). In recent years,
well-controlled animal models have demonstrated that phages can rescue animals
from a variety of fatal infections, while non-controlled clinical reports
published in
Key words: bacteriophage; phage; bacterial viruses; bacterial infections;
multidrug resistance.
Phages are a kingdom of viruses that infect bacteria, and are distinct from the
animal and plant viruses. Phages can have either a “lytic” or a “lysogenic”
life cycle. The lytic phages are the most suitable candidates for phage
therapy, because they quickly reproduce within and lyse the bacteria in their
host range, growing exponentially in number in the process. Depending on
the species and conditions, each “parent” phage can produce on average
approximately 200 “daughters” per lytic cycle. If each daughter infects and
kills a host bacterium there will be 40 000 progeny at the end of the 2nd
cycle; 8 million at the end of the 3rd cycle; 1.6 billion at the end of the 4th
cycle; and so on.
Some practitioners used phages as therapeutic agents in the West, from the
1920s to the early 1950s (referred to hereinafter as the “historic era”). This
review will describe: 1) some of the key reasons this form of therapy failed to
take root in the West; 2) its previous and current use in some enclaves of
Eastern Europe; 3) recent animal models which suggest that phage therapy might
be useful for humans; 4) the fact that the emergence of antibiotic-resistant
infections has
opened a second window of opportunity for phage therapy; and 5) the advantages
that might be gained by administering phages along with antibiotics, as a
combination therapy.
Past History
General
information
A number of reviews provide details on phage therapy’s ascent and decline in
the historical era 1–3, 13, 14.
We will summarize some of the more salient features
of this history.
Phages were discovered in 1915 by British microbiologist Felix Twort, and,
independently in 1917, by French-Canadian microbiologist Felix d’Hérelle. Twort
did not pursue his discovery, whereas d’Hérelle systematically investigated the
nature of bacteriophages and explored their ability to function as therapeutic
agents 5, 6.
D’Hérelle received a fair measure of fame for his discovery. He was appointed
Professor of Protobiology at
There are many attributes of phages (see Table 1) that would tend to favor a
positive outcome in therapy. Despite these attributes of phages, there were so
many problems with the way phage therapy was practiced in the historical era
that, by the time antibiotics were introduced in mid-century, it was already in
sharp decline in the West. The investigators who developed antibiotics did not
make the kinds of mistakes exhibited by the early phage investigators.
Key problems with phage therapy, and how the problems can be
overcome
Problem 1. Host range
The issue. Phages tend to have a relatively narrow host range, posing certain
disadvantages. A disadvantage is that one should administer only those phage
strains shown to be strongly lytic for the bacterial strain infecting the given
patient. If the patient’s condition is too critical to take the time required
for this matching, then one should use a grouping (a panel) of phages,
where each of the phages therein has a broad-enough host range that most
strains of the bacterial target are likely to be targeted. In his lectures to
the New York Academy of Medicine in 1931, d’Hérelle cited the reports of other
colleagues whose initial trials used phages “off the shelf” (without being
shown to be virulent for the bacteria infecting the patient) and had negative
outcomes, but who did match the phage to the bacteria in subsequent trials and
obtained positive outcomes.
The solution. 1) Screen the bacteria infecting a given patient against a panel
of phages, to ensure that one of the phage strains will be lytic (analogous to
the “culture and sensitivity test” that physicians should perform; and 2)
develop “multivalent” phages that lyse all or most of the bacterial strains
within a given species of pathogen.
..........................................................................................................................
Table 1.
Attributes of
phages that tend to favor a therapeutic response
The issue: Limitations of antibiotics Advantages of phages
Fate of the “drug” molecule
Concentration of the “drug” required to kill a given bacterium within the
spectrum
Ability to overcome bacterial resistance
Spread of bacterial resistance
Metabolic destruction of the molecule, as it works
Numerous molecules of the antibiotic are needed to kill a given bacterium.
During initiation of
therapy (and between doses), the sub-lethal dose that bacteria “see” affords
them the opportunity to express resistance genes. Antibiotics are fixed,
immutable chemicals that cannot adapt to a bacterial mutation and therefore
become obsolete. Bacteria that have resisted them can pass along the resistance
trait within and between species.
The antibiotics in use tend to be broad spectrum, thereby provoking resistance
in several species and genera of bacteria (in addition to the one targeted) .
Exponential growth in numbers, so that the “drug” makes more of itself at the
site of infection, where it is needed.
“All or nothing” effect: one phage particle is sufficient to kill a given
bacterium.
Phages are “living” organisms that undergo
mutations, some of which can overcome
bacterial mutations. E. g., mutated phage tail
fibers can allow binding to a mutant bacterial
receptor, or mutated phage DNA can escape
cleavage by mutant bacterial endonucleases
Although there are some exceptions, phages tend
not to cross species boundaries. Thus even
though the targeted bacterial species may
become resistant to the phage, it is unlikely that
other species will
268 R. M. Carlton: Phage Therapy in the Past and Future
....................................................................................................................
Problem 2. Bacterial debris
present in the phage preparations
The issue. Injection of even minute amounts of endotoxin and other bacterial
debris can be fatal to patients. Unfortunately, many of the phage preparations
used by practitioners in the historical era were crude lysates. When these
preparations were injected i.v., i.p., and in some cases even intrathecally,
any beneficial effect of the phages would likely have been counteracted
by illness and deaths resulting from the endotoxin.
The solution. Modern technology allows density centrifugation, banding, and
other methods of purification.
Problem 3. Attempts to remove host bacteria from
therapeutic preparations
The issue: In order to ensure that phage preparations would not contain live
bacteria, some early investigators added mercurials and/or oxidizing agents,
while others heated them. It is now known that such agents and procedures will
denature or otherwise inactivate the phage coat proteins. These investigators
did not check for continued viability of the phages. The false-negative results
of such studies were the unintended (but inevitable) consequence of such
practices.
The solution: Sterile filtration. If chemical agents must be used, retitrate
the preparation over time to ensure that the phage remain viable.
Problem 4. Rapid clearance of phages
The issue. In fairness to phage investigators in the historical era, at the time
it was not an accepted practice, in any discipline, to conduct pharmacokinetic
studies. However, had the early phage investigators conducted such studies,
they would have discovered that bacteriophages (being foreign proteins) tend to
be rapidly cleared from the circulation. This clearance
problem was first documented by Merril and his colleagues in 1973 who injected
high titers of phage lambda into non-immune germ-free mice. They discovered
that the phages were rapidly cleared by the spleen, liver and other filtering
organs of the reticulo-endothelial system (RES)7. This was a seminal
observation, given Gunther Stent’s widely-accepted statement that one of
the principal reasons phages had failed as a therapeutic was their supposed
inactivation by pre-existing antibodies to them. However, any clearance of the
phages from the bloodstream of the germ-free animals used by Merril and his
group (ref.7) would not be due to antibodies,
since those animals had never previously been exposed to bacteria or bacteriophages
(and so would not have antibodies). Moreover, the phages in Merril’s experiment
remained viable in the spleens of these animals over a period of several days,
indicating that they were neither neutralized by antibody nor engulfed by
macrophages. Rather, they appeared to have been passively entrapped in
(sequestered by) these filtering organs. Such trapped phages would be
unavailable to reach bacteria.
The solution. The author of this review collaborated with investigators at the
U.S. National Institutes of Health (MERRIL et al.11) in the development of a
method to isolate and amplify phage strains that are cleared at a slower rate.
We reasoned that in all species of phage,
minor variations in coat proteins might be present that would enable some variants
to be less easily recognized by the RES organs and to thereby remain in the
circulation for longer periods of time than the “average” wild-type phage. In
this “serial passage” method, the wild-type preparation is injected into an
animal, and then blood samples are taken at progressively longer
time points. Any phages found in the blood sample are grown to high titer and
reinjected. Through iterative rounds of passage, one can amplify the
long-circulating strains being isolated.
For coliphage lambda as well as for salmonella phage P22,
phage variants were isolated in this manner that were much longer-circulating
than the wild-type. For example, for every 100 000 particles of the wild-type
lambda used at baseline, only one particle remained in circulation at 18 h;
whereas for the long-circulating phage mutant isolated at the 8th round of
serial passage, for every 100 000 injected, at 18 h 62 500 particles remained
in circulation. For each moment of time, far more of these long-circulating
phages are propagating exponentially, as compared to the situation for the
wild-type phages.
As predicted, these long-circulating phages were far superior to the wild-types
from which they were derived, in terms of rescuing animals from an
otherwise-fatal fulminant bacteremia: 1) with no treatment, all animals were
dead within 48 h; 2) treatment with the wild-type phages prevented death, but
the animals became critically ill (a human with such degrees of illness would
be in the intensive care unit); and 3) in contrast, with administration of the
long-circulating phage strain, the only sign of illness seen was mild lethargy.
These results were published in the Proceedings of the National Academy of
Sciences (ref. 11), and were accompanied by a Commentary by Nobel laureate Dr.
JOSHUA LEDERBERG 8.
R. M. Carlton: Phage Therapy in the Past and Future P 269
We have elucidated the molecular basis of the mutation in lambda that reduced its rate of clearance:
a single point mutation, an A to G transition, had occurred in the gene encoding the major head protein E.This mutation substituted a basic amino acid (lysine) for an acidic one (glutamic acid), causing a double charge shift readily seen on 2D gel electrophoresis.Computer modeling predicted that the mutation occurred in a loop of the E protein that sticks out into space and that therefore may interact with the external environment. A double charge shift in this region ofa protein that is highly represented on the surface of the virion could conceivably alter the phage’s interaction
with the microcirculation of the spleen, in such a waythat the mutant phage is less easily entrapped than thewild-type.
Problem 5. Lysogeny
The issue. It was not until the late 1950s that Lwoff demonstrated the ability of some phage genomes to integrate into the bacterial chromosome as “prophages.”After a period of time (up to days or weeks, or longer), such prophages can enter the lytic cycle, and will thus appear as plaques on a bacterial lawn. It is likely that some phage therapy trials in the historic era had a negative outcome due to the inadvertent use of phage strains that, being lysogens, could not provide the rapid lysis and exponential growth in numbers that are needed for full efficacy.
The solution. Use only phages that are lytic; sequence phages that are strong candidates for clinical
trials, looking for (among other things) homologies to known genes of lysogeny.
Problem 6. Anti-phage antibodies
The issue. There are reports in the literature20 that neutralizing antibodies appear a few weeks after administering phages to humans or animals. Given the time lag, antibodies would not seem likely to interfere with an acute treatment lasting a week or so. However, in chronic treatment, or in treatment of a recurrence of the same bacterial infection, the neutralizing antibodies might prevent some proportion of the administered dose of phages from being able to adhere to the bacterial target.
The solution. In treating chronic or recurrent infections it may be possible to administer a higher dose of phage, to compensate for those that are rendered non-viable by interaction with neutralizing antibodies. In any case, the types and titers of antibodies that develop should be systematically studied in humans.
Problem 7. Failure to establish scientific proof of efficacy In scholarly reviews of comparative styles of research, Dutch historian TON VAN HELVOORT24 has discussed d’Hérelle’s systematic failure to conduct double-blind studies. As van Helvoort pointed out, while it is true that ethical problems are faced by anyone who has to administer placebo to some patients (in order to prove efficacy), nevertheless the investigators who later tested antibiotics did conduct double-
-blind, placebo-controlled trials. Van Helvoort points out that, even when using phages to treat an epidemic of diarrhea in poultry on a French farm, d’Hérelle failed to use a placebo on half the flock (a situation where ethical considerations would not have been an issue). As a consequence, all reports of phage therapy’s successes in the historical era were anecdotal. No systematic proof was available to demonstrate that the results were reliable and repeatable.
Problem 8. The scientific style of phage investigators in the historical era, D’Hérelle’s failure to conduct placebo-controlled studies, even on chickens, is an important example of his style. This story is a notable example of the negative impact an investigator’s personality can have on the outcome of a discovery, and d’Hérelle’s style contrasts sharply to the strongly positive influence that other scientists (such as Pasteur) have had on the outcomes of their discoveries. Whereas Pasteur excelled at
conceiving of definitive experiments, and was persuasive in style, d’Hérelle failed to conduct definitive experiments, and was antagonistic rather than persuasive. For example, d’Hérelle maintained to the end that phages are the sole mechanism of defense against bacterial infection. While he may have been correct in his view that epidemics can sometimes be checked by the spontaneous appearance of a lytic strain of phage, nevertheless he was incorrect in categorically dismissing the discoveries of Nobel laureates Metchnikoff and Ehrlich, who had shown that cellular elements (white blood cells) and humoral elements (antibodies and complement) constitute the innate host defenses against infection. D’Hérelle was afforded many opportunities to integrate his discovery with those of Metchnikoff and
Ehrlich, but refused to the end (see below).
In addition to the damage he was doing to himself and his cause with this adamance, d’Hérelle was attacked by Nobel laureate Jules Bordet (for whom Bordetella pertussis was named), who had an intense dislike not just for d’Hérelle’s science but also for the man himself. Bordet used his considerable influence to discredit D’HÉRELLE 5.
R. M. Carlton: Phage Therapy in the Past and Future P 270
D’Hérelle retreated from attacks by Bordet and others, and moved to Soviet Georgia in the 1930s (see ref. 13). An ardent communist, he dedicated the last of his published treatises to Josef Stalin. He was in
Animal Models of Phage Therapy
From the 1950s to the 1980s there was little published on the subject of phage therapy. Then papers
began to appear demonstrating the utility of phage therapy in animal models. For example, phages were shown to be effective in rescuing rats from fatal systemic infections (induced with E. coli)14 in rescuing calves and lambs from fatal diarrhea (induced with E. coli)15, 16, in rescuing chicks from fatal diarrhea (induced with S. typhimurium) 4, and in preventing destruction of skin grafts in burned rabbits by Pseudomonas aeruginosa18. As mentioned above MERRIL et al.11 demonstrated in
1996 that mice with fulminant E. coli bacteremia could be rescued by phages, and that long-circulating phage variants were superior to the wild-types (see below). In one of those studies cited, Smith and Huggins (ref. 6) demonstrated that, in rats inoculated with a lethal intramuscular dose of E. coli, a single injection of a phage preparation was more effective than multiple injections of antibiotics (chloramphenicol, tetracycline, etc.). This work was replicated in 1997 by LEVIN and BULL9, who used mathematical modeling in a population dynamics approach to study the titers of phages and bacteria in the animals. The investigators concluded that the reason a single injection of phage was
superior to multiple injections of antibiotics was that the phages grew exponentially in number, overwhelming the bacteria present.
Current Status of Human Phage Therapy Efforts
disease progression. The bacterial pathogens targeted included Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and E. coli. The phages used by these investigators are reported to have cured approximately 90% of the cases.
The criteria of cure were cessation of suppuration and,
where applicable, complete closure of wounds/fistulae
(many of which had been draining for months).
These investigators administer phages orally, because
they are aware of the hazards of administering
them parenterally (not all of the bacterial debris has
been removed). They pre-treat the patients with antacids
and gelatin in order to protect the phages from
destruction by gastric acidity. These same investigators
have published evidence that phages administered
orally to humans in this manner do in fact reach the
bloodstream26.
The Polish investigators have been rigorous in
matching the phages to the bacterial strain infecting the
given patients. Their practice, as stated in the published
reports, is to culture the bacteria during the course of
treatment, so that the occurrence of a mutant resisting
the phage can be countered by switching to a different
phage strain. The group also has panels of multivalent
phages available, for use in fulminant infections (such
as septicemia with acute respiratory distress syndrome)
where time is insufficient to classify the offending bacteria
or to match phages to bacteria.
The group now has statistics on the treatment of
approximately 1 300 cases. The overall cure rate across
the spectrum of pathogens and sites of infection is approximately
86% (personal communication from Dr.
B. Weber-Da˛browska).
A criticism of the work by S´ lopek’s group is that
* The push of the German army into the region of Georgia was
intended not only to capture the region’s oil wells, but also to obtain
the collection of phages manufactured at the Eliava-d’Hérelle Institute
in Tblisi. That institute was providing phages to the Russian
army, to control dysentery, Staphylococcus aureus infections of
wounds, and other bacterial problems associated with war.
R. M. Carlton: Phage Therapy in the Past and Future 271
the absence of placebo controls means the power of
suggestion cannot be definitively ruled-out. It is clear
that the difficulties of that nation’s economy over recent
decades has denied the investigators the financial resources
needed to enroll matched cohorts in a placebo
arm of a clinical trial. While the criticism is valid, and
absolute proof of principle can be obtained only
through placebo-controlled trials, nevertheless the usefulness
of the data is improved by the detailed statistical
accounting of the percentages of complete, partial and
nil response. One of the factors that enables this author
to find the data from Poland more believable (even in
the absence of double-blind proof) is that in conditions
such as emphysema where phage efficacy might be
somewhat impeded, the group’s statistics show that the
success rate is considerably lower than for other conditions
where such impediments do not obtain*.
The
in the 1930s by d’Hérelle and his Georgian colleague,
Eliava, continues to this day. In the 1970s, under the
direction of Dr. Teimuraz Chanishvili, the Eliava-d’Hérelle
Institute had a large staff manufacturing considerable
quantities of phage preparations per year, primarily
for the control of dysentery in the troops of the Soviet
Army. This group has anecdotal evidence of the efficacy
of phage therapy. They report, for example, that
in certain adult and pediatric hospitals it is routine for
their phage preparations to be administered topically on
surgical incisions. Given the lack of statistical analysis,
there is little to be said other than the anecdotal reports
are encouraging that phage therapy can be useful.
Multidrug-Resistant (MDR) Bacteria Have
Created a Need for Phage Therapy
Several species of bacteria have become resistant to
most antibiotics, with some strains being resistant to all
antibiotics. One example is vancomycin-resistant Enterococcus
faecium (VRE), a low-virulence pathogen
that now frequently causes fatal bacteremias due to
complete resistance2. Another example is vancomycin
intermediate-resistant Staphylococcus aureus (VISA),
strains of which have recently emerged in three nations
(
killed 4 patients to date. Such strains spread throughout
Japanese hospitals within a year of their first appearance.
Unfortunately, it has been demonstrated that some
hospital strains of methicillin-resistant S. aureus (MRSA)
that are widespread have become vancomycin resistant
upon exposure of the patients to vancomycin1, 2. Experts
predict that S. aureus will progress to become
completely resistant to vancomycin (the antibiotic of
last resort for most strains of this pathogen), and that
when this occurs, millions of people will die each year
from infections that had until recently been fairly easy
to control. Based on such developments and impending
developments with pathogens such as MRSA and VRE,
opinion leaders have been warning that we are entering
the “Post-Antibiotic Era”.
While pharmaceutical companies are developing
new antibiotics to counter the trend, it has been shown
that half a century of global antibiotic abuse has
equipped the surviving bacteria with “supergenes” that
enable them to quickly resist new classes of antibiotics,
even those to which they have never been exposed1.
Examples of the “supergenes” are mutations that 1) enable
bacteria to pump out several classes of antibiotics
(through an efficient efflux pump), or that 2) alter the
antibiotic binding sites on ribosomal subunits, so that
several different classes of antibiotics can no longer
inhibit those subunits. As a consequence, in recent
years, by the time newer antibiotics have gone through
clinical trials and have reached the market, 20% or
more of clinical isolates in the hospitals are already
resistant to them at the time of regulatory approval, and
within a few more years the majority of strains are resistant.
Future Prospects for Phage Therapy
Infectious disease experts have warned that there is
now a compelling need to develop totally new classes of
antibacterial agents, ones that cannot be resisted by the
same genes that render bacteria resistant to antibiotics.
Phage therapy represents such a “new” class. We
believe that the impediments cited above (bacterial debris
in the preparations, rapid clearance in the body,
etc.) can be overcome, freeing up the phages so that
their attributes (such as exponential growth, and the
ability to mutate against resistant bacteria) can be used
to great advantage.
There are 3 additional attributes of phages that
should be noted:
Host specificity. While the host specificity is somewhat
of a drawback (requiring a matchup of phage to
bacterial target, and/or the development of highly
multivalent phages), it also offers the great advantage
that the phages will not kill other species of bacteria.
* Conditions where phage efficacy is predicted to be reduced
would include 1) hypoxic sites, where bacterial replication is slower
and therefore phage replication is reduced; and 2) chronic obstructive
pulmonary disease, where high acidity and proteases would be
expected to inactivate some percentage of the phages.
272 R. M. Carlton: Phage Therapy in the Past and Future
Thus, e.g., phage therapy is not likely to kill off the
healthy flora of the intestines, lungs or urogenital tract,
and it is therefore unlikely to provoke the illnesses and
deaths seen when antibiotics cause overgrowth of pathogens
(such as Clostridia difficile and Candida albicans).
Genetic engineering. It is possible to genetically engineer
phages to express new traits of potential value.
In so doing, scientists will have to deal with the legitimate
concerns of regulatory agencies concerning recombinant
organisms. The regulatory obstacles may be
well worth the price, given the powerful engineering
tools that are currently available.
Ideal candidates for co-therapy with antibiotics. If
a given bacterium acquires resistance to a phage (e.g.
by a mutation in the receptor site or in the endonuclease
enzymes), that mutation is not likely to “teach” the
bacterium to resist the antibiotics (which do not target
those structures). Similarly, if a given bacterium acquires
resistance to an antibiotic (e. g. by a mutation in
the reflux pump or in the ribosomal subunits), that mutation
is not likely to “teach” the bacterium to resist the
phage (which does not target those structures). Thus, if
the bacterium is exposed to both agents, the odds are
remote that any resistance genes it starts to express (or
acquires anew) will enable it to survive. There are reports
that bacteria tend to mutate against antibiotics
once in every 106 divisions, while they tend to mutate
against phages once in every 107 divisions. Therefore
the odds of a given bacterium mutating against a phage
and an antibiotic at the same time would be the product
of 106×107, meaning it would likely take 1013 bacterial
divisions for such a double mutation to occur. Given
that low probability, the co-administration of phages
and antibiotics may help prevent the emergence of bacterial
resistance to antibiotics, thereby greatly prolonging
their clinical usefulness (and vice versa). Just as
multiple classes of anti-HIV medications are administered
to AIDS patients, to prevent the emergence of
resistant strains of that virus, so it is that co-therapy
with phages and antibiotics may also prove to be of
great clinical value.
Conclusion
Multidrug-resistant bacteria have opened a second
window for phage therapy. Modern innovations, combined
with careful scientific methodology, can enhance
mankind’s ability to make it work this time around.
Phage therapy can then serve as a stand-alone therapy
for infections that are fully resistant. It will also then
be able to serve as a co-therapeutic agent for infections
that are still susceptible to antibiotics, by helping to
prevent the emergence of bacterial mutants against
either agent.
References
1. ACKERMANN H. -W. and DUBOW M. (1987): Viruses of prokaryotes
I: General properties of bacteriophages (chapter 7). Practical
applications of bacteriophages. CRC Press,
2. ALISKY J. et al. (1998): Bacteriophages show promise as antimicrobial
agents. J. Infect., 36, 5–15.
3. BARROW P. A. and SOOTHILL J. S. (1997): Bacteriophage therapy
and prophylaxis: rediscovery and renewed assessment of
the potential. Trends Microbiol., 5, 268–271.
4. BERCHIERI A. et al. (1991): The activity in the chicken alimentary
tract of bacteriophages lytic for Salmonella typhimurium.
Res. Microbiol., 142, 541–549.
5. D’HÉRELLE F. (1917): Sur un microbe invisible antagoniste des
bac. dysentériques. Crit. Rev. Acad. Sci. Paris, 165, 373.
6. D’HÉRELLE F. (1922): The bacteriophage: its role in immunity.
Williams and Wilkens Co. /Waverly Press,
7. GEIER M., FRIGG M. E. and MERRIL C. (1973): Fate of bacteriophage
lambda in non-immune germ-free mice. Nature, 246,
221–222.
8. LEDERBERG J. (1996): Commentary. Proc. Natl. Acad. Sci.
USA, 93, 3167–3168.
9. LEVIN B. and BULL J. J. (1996): Phage therapy revisited: the
population biology of a bacterial infection and its treatment
with bacteriophage and antibiotics. Am. Naturalist, 147, 881–898.
10. LEVY S. (1992): The antibiotic paradox. Plenum Press,
York
11. MERRIL C. et al. (1996): Long-circulating bacteriophage as
antibacterial agents. Proc. Natl. Acad. Sci. USA, 93, 3188–
2192.
12. MURRAY B. (1998): Diversity among multidrug-resistant Enterococci.
Emerging Infect. Dis., 4, 37–47.
13. SHRAYER D. (1996): Felix d’Hérelle in
94, 91–96.
14. SMITH H. W. and HUGGINS R. B. (1982): Successful treatment
of experimental E. coli infections in mice using phage: its general
superiority over antibiotics. J. Gen. Microbiol., 128, 307–318.
15. SMITH H. W. and HUGGINS R. B. (1983): Effectiveness of
phages in treating experimental E. coli diarrhoea in calves, piglets
and lambs. J. Gen. Microbiol., 129, 2659–2675.
16. SMITH H. W. and HUGGINS R. B. (1987): The control of experimental
E. coli diarrhea in calves by means of bacteriophage.
J. Gen. Microbiol., 133, 1111–1126.
17. SMITH T. et al. (1999): Emergence of vancomycin resistance in
Staphylococcus aureus. N. Engl. J. Med., 340, 493–501.
18. SOOTHILL J. S. (1992): Treatment of experimental infections of
mice with bacteriophages. Med. Microbiol., 37, 258–261.
19. SUMMERS W. C. (1998): D’Hérelle. Yale University Press (in
press).
20. S´LOPEK S. and KUCHAREWICZ-KRUKOWSKA A. (1987): Immunogenic
effect of bacteriophage in patients subjected to phage
therapy. Arch. Immunol. Ther. Exp., 35, 553–561.
21. S´LOPEK S., KUCHAREWICZ-KRUKOWSKA A., WEBER-DA˛BROWSKA
B. and DA˛BROWSKI M. (1985): Results of bacteriophage
treatment of suppurative bacterial infections. IV. Evaluation of
R. M. Carlton: Phage Therapy in the Past and Future 273
the results obtained in 370 cases. Arch. Immunol. Ther. Exp.,
33, 219–240.
22. S´LOPEK S., KUCHAREWICZ-KRUKOWSKA A., WEBER-DA˛BROWSKA
B. and DA˛BROWSKI M. (1985): Results of bacteriophage
treatment of suppurative bacterial infections VI. Analysis of
treatment of suppurative staphylococcal infections. Arch. Immunol.
Ther. Exp., 33, 261–273.
23. S´LOPEK S., WEBER-DA˛BROWSKA B., DA˛BROWSKI M. and KUCHAREWICZ-
KRUKOWSKA A. (1987): Results of bacteriophage
treatment of suppurative bacterial infections in the years 1981–
1986. Arch. Immunol. Ther. Exp., 35, 569–583.
24. VAN HELVOORT T. (1992): Bacteriological and physiological
research styles in the early controversy on the nature of the
bacteriophage phenomenon. Med. Hist., 36, 243–270.
25. WALDVOGEL F. (1999): New resistance in Staphylococcus
aureus. N. Engl. J. Med., 340, 556–557.
26. WEBER-DA˛BROWSKA B., DA˛BROWSKI M. and S´ LOPEK S.
(1987): Studies on bacteriophage penetration in patients subjected
to phage therapy. Arch. Immunol. Ther. Exp., 35, 563–568.
Received in May 1999
Accepted in June 1999
274 R. M. Carlton: Phage Therapy in the Past and Future
