The prospect for bacteriophage therapy in Western medicine.(Report)
Author's Abstract:
Bacteriophage (phage) have been used for clinical applications since their
initial discovery at the beginning of the twentieth century. However, they have
never been subjected to the scrutiny -- in terms of the determination of
efficacy and pharmacokinetics of therapeutic agents -- that is required in
countries that enforce certification for marketed pharmaceuticals. There are a
number of historical reasons for this deficiency, including the overshadowing
discovery of the antibiotics. Nevertheless, present efforts to develop phage
into reliable antibacterial agents have been substantially enhanced by
knowledge gained concerning the genetics and physiology of phage in molecular
detail during the past 50 years. Such efforts will be of importance given the
emergence of antibiotic-resistant bacteria.
Full Text: COPYRIGHT 2003 Nature Publishing Group
Author(s): Carl R. Merril [1]; Dean Scholl [1]; Sankar L. Adhya [2]
The first widely used antibiotic, penicillin, was discovered 75 years ago.
Since then, the production of antibiotics has grown into an industry with a
market value estimated to exceed US $25 billion per year. Although antibiotics
have saved countless lives, their widespread use has contributed to an increase
in the incidence of antibiotic-resistant bacterial strains. For example, at the
beginning of the antibiotic era Streptococcus pneumoniae was highly
sensitive to penicillin, yet now, in some regions of the world, 25% of S.
pneumoniae strains are resistant [1]. In addition, 70% of hospital-acquired
bacterial infections in the United States are now resistant to one or more of
the main antibiotics [2] and greater than 50% of clinical Staphylococcus
aureus isolates in Japan are now multidrug resistant [3]. However,
antibiotics are only one example of antibacterial entities that have arisen
through the evolutionary competition between other species and bacteria.
Another entity, the bacterial viruses (bacteriophage or PHAGE), might provide
an alternative to fill the space created in the physician’s toolkit left by the
dearth of new medicines to combat infections with antibiotic-resistant bacteria
(Fig. 1).
The ability of phage to replicate exponentially and kill pathogenic strains
of bacteria indicates that they should play a vital role in our armamentarium
for the treatment of infectious diseases. In fact, such an application for
phage was apparent to Felix d’Herelle after they were discovered by Twort in
1915 (Refs 4,5). d’Herelle was encouraged by his early experiments, in which he
used phage to treat avian typhosis in chickens, shigella dysentery in rabbits
and humans with bacillary dysentery. Following the reported successes of these
experiments, d’Herelle travelled around the world stimulating basic and
clinical phage research. Inspired by d’Herelle, the British medical officer
Lieutenant Colonel Morison used phage for the prophylactic treatment of cholera
epidemics in the Naogaon region of
d’Herelle was also important in the establishment of a phage institute in
There was also an interest, in the 1920s and 1930s, in phage therapy
in the
Re-examination of potential efficacy
Despite the chequered history of phage
therapy, concerns over the increased
incidence of antibiotic resistance has led investigators to examine the
possibility of developing phage therapy into a reliable clinical tool. In one
recently published animal study, a vancomycin-resistant Enterococcus faecium
strain was used to induce a fatal bacteraemia (within 48 hours) in mice.
Treatment with a single intraperitoneal injection of phage 45 minutes after the
bacterial challenge was sufficient to rescue 100% of the animals, and even when
treatment was delayed until the animals were moribund, phage administration was
able to rescue 50% of the mice [21]. Similar results have been obtained using
phage to treat animals infected with methicillin-resistant S. aureus
[22]. In addition, phage have been used to treat Acinetobacter baumanii
or Pseudomonas aeruginosa and animals with local and systemic disease
caused by Vibrio vulnificus [23, 24].
Phages are also capable of treating antibiotic-resistant intracellular
pathogens. Broxmeyer et al . [25] have demonstrated that it is possible
to use Mycobacterium smegmatis , an avirulent mycobacterium, as a vector
to deliver the LYTIC PHAGE TM4 to treat intracellular mycobacterium infections
(with either Mycobacterium avium or Mycobacterium tuberculosis )
in macrophages. Phages have also been effective in the treatment of
non-systemic infections, such as gastrointestinal infections caused by
enteropathogenic strains of Escherichia coli in calves, pigs and lambs
and ileocecitis caused by Clostridium difficile in hamsters [26, 27, 28,
29]. Phages have also been shown to be effective in preventing the destruction
of skin grafts by P. aeruginosa [30]. Terrestrial animals are not the
only candidates for phage therapy, as recent studies have shown that phage
can be used to treat bacterial diseases of fish in aquiculture [31], bacterial
blight of geranium [32] and bacterial spot on tomatoes [33].
Phage pharmacokinetics
Although phage therapy has a long
history, pharmacokinetic data are still rudimentary. Most clinical applications
of phage therapy
have relied on the oral administration of phage preparations. This choice
offers the possibility of reducing side effects from contaminants, including
ENDOTOXINS and EXOTOXINS, but it might not provide the most effective
therapeutic regimen(s) [34]. Some early practitioners of phage therapy
recognized the need for pharmacokinetic information. The first effort to obtain
such data in 1921 led to the observation that phage injected into the
circulatory system of rabbits could still be found in the spleen long after the
elimination of phage from other organs or the blood [35]. This finding was
corroborated by both qualitative and quantitative experiments, in which a
decrease in phage titre in the blood of mice injected with a Staphylococcus
phage (a decrease of four orders of magnitude of phage titres was observed 5
minutes after intravenous injection, and by seven logs two hours later) was
attributed to elimination by the reticulo-endothelial system (RES) [17, 36].
The relative roles of the liver and spleen in this process were determined
by using 51 Cr-labelled phage. In these experiments the liver
phagocytosed more than 99% of the phage in the circulatory system and
inactivated the phage at a higher rate than the spleen [37]. Antibodies
associated with the ADAPTIVE IMMUNE SYSTEM were not involved in this process,
as demonstrated by experiments using germ-free mice that had no detectable
antibodies to the phage strain used. Systemically administered phages were
rapidly eliminated from the circulation of the germ-free mice. Furthermore,
these quantitative experiments demonstrated that the oral administration of
phage was not an effective method for delivery to systemic sites [34] (Fig. 2).
Recognition that the RES can remove a significant proportion of administered
phage led to the development of a serial passage selection method to isolate
phage mutants with a greater capacity to remain in the circulatory system of
the mouse. These ’long-circulating’ phage mutants also proved to be more
effective therapeutically [38].
In studying the pharmacokinetics of phage it should be noted that phage DNA,
like any other foreign DNA, can get into mammalian cells and, on rare
occasions, into chromosomes [39, 40, 41]. There have also been reports of
phage-induced enzyme activity in mammalian cells, albeit at low levels,
following exposure to phage or phage DNA [42, 43]. Although there are efforts
underway to enhance the capability of phage to serve as vectors for targeted
gene delivery in mammalian cells [44], there is evidence that in the natural
setting such effects are normally minimal: phages are, for example, associated
with bacteria in our colon, nose, throat and skin throughout our life span.
For nearly all pharmacological agents, information on drug distribution and clearance
would be sufficient for pharmacokinetic studies. However, unlike most
pharmaceuticals, phage can replicate exponentially. The exploitation of phage
as antibacterial therapeutics requires knowledge of three dynamic components:
the infected human, the infecting bacteria and the phage, and their complex
interactions. Of these three dynamic components, two of them -- the bacteria
and the phage -- are capable of exponential growth during the course of an
infection and its treatment. Given this situation, it is crucial to ensure that
the phage titres employed are sufficient for a successful therapeutic outcome
(Fig. 3). To explore such parameters, Smith and Huggins used two experimental
models, one in which mice were infected intracerebrally with E. coli K1
and another in which they were infected intramuscularly. Phage was administered
intramuscularly in both of these studies. The phage levels were found to be
highest in the infected tissues and they fell as the bacterial levels in the
infected tissue decreased.
These results corroborated the earlier finding of Dubos et al . [18]
that mice infected intracerebrally with Shigella dysenteriae were
rescued by administering phage into the peritoneal cavity [45]. In these
experiments, the survival of untreated animals was 3.6%, whereas the survival
of phage-treated animals was 72%. In addition, phage levels were observed to
increase at the site of the infection -- the brain -- whereas the blood levels
of phage seemed to be a "reflection of the events occurring in the
brain". In uninfected control animals injected with phage, phage levels in
the blood were compatible with the dilution of the phage in the total blood
volume of the mouse. These researchers also showed that heat-inactivated phage
provided no protective effects unless given days before the bacterial
infection, and suggested that the protective effect of such heat-inactivated
phage might result from the activation of antibacterial immunity by bacterial
products present in the phage LYSATE.
Levin and Bull [46], and Payne and Jansen [47], have developed formal
mathematical models on the basis of data from the Smith and Huggins [26] study.
The Payne and Jansen [47] model included terms for the loss of phage resulting
from interaction with mammalian systems, such as the RES. Their analysis with
this model corroborated the suggestion by Alinsky et al . [8] that
antibiotic and phage therapies might not be synergistic. Although such models
are important for pharmacological planning, present efforts are based on limited
datasets. Recently developed methods of visualizing bacterial infections in
live animals using bioluminescent strains of bacteria might help in such
pharmacokinetic studies [48]. This method uses a high sensitivity
charge-coupled device camera to monitor bacteria in live mice. The bacteria are
made bioluminescent by incorporating a luciferase transposon cassette into
their genome. As it is feasible to incorporate luciferase transposon cassettes
into phage genomes, it should also be possible to follow phage interactions
with bacterial infections in vivo .
Phage immunogenicity
Phage immunogenicity is important for two reasons: first, because of
potential adverse reactions, such as anaphylactic shock, and second, because of
its effects on the pharmacokinetics of phage
therapy. When phage are first used, some
behave as NEO-ANTIGENS, as there are no pre-existing antibodies for these
phage. This feature, along with the lack of apparent side effects, has
permitted the use of phage [phi]X174 as a probe of human and animal immune
sytems [49, 50, 51]. Despite the apparent lack of antibodies to phage that
present as neo-antigens they still interact with the innate immune system, as
demonstrated by the rapid loss of phage injected into the circulatory system of
germ-free mice [34] and the capacity of the liver to phagocytose more than 99%
of administered phage within 30 minutes of inoculation [37]. It is important to
note that not all phage present as neo-antigens, as Kucharewica-Krukowska and
Slopek noted in a study in which 11% of their healthy controls and 23% of their
patients had antibodies against a Staphylococcus phage strain before
administration of phage therapy [45].
If a particular phage strain were used repeatedly as an therapeutic
antibacterial agent, in addition to the interactions with the innate immune
system, the adaptive immune system would be stimulated and result in the
production of antibodies. Such activation of the adaptive immune system relies
on somatic mutations and clonal expansion of T and B cells, which can take at
least three to five days. In normal individuals injected with the highly
immunogenic phage [phi]X174, the phage is normally cleared within three days
and a primary immunoglobulin M (IgM) response can be observed that peaks two weeks
after the initial injection or immunization. If another injection is made six
weeks later, the IgM and IgG antibody titres increase and peak within one week
of the second injection; subsequent phage injections result in further
increases in the IgG titres [50]. Patients with severe combined immune
deficiency (SCID), which is characterized by the absence of both B and T cell
functions, display a prolonged period for the clearance of phage, with phage
present up to four to six weeks after the initial injection. In addition, SCID
patients do not develop detectable antibody responses to phage, even after
repeated injections. Ochs and his colleagues have also found that although
[phi]X174 phage is a potent antigen, it causes no recognized toxic effects in man
[49, 52].
Similar findings were reported for the phage ENB6 used to treat mice
infected with vancomycin-resistant Enterococcus [21]. To determine the
immunogenicity of this phage, mice were given monthly injections. After the
third in a series of five monthly injections, the titres of IgG and IgM
increased 3,800-fold and fivefold, respectively, above background. The IgG
levels did not change substantially after the third phage injection. No
anaphylactic reactions, changes in core body temperature or other adverse
events were observed in the mice over the course of these multiple injections
of phage [21]. It might be possible to develop phage that are less antigenic by
using phage-displaying peptide libraries or affinity matrixes made up of
antibodies from human serum. This type of approach has been initiated to
decrease the immunogenicity of therapeutically important enzymes [53].
Bacterial host specificity of phage
The bacterial host range of phage is generally narrower than that found in
the antibiotics that have been selected for clinical applications. Most phage
are specific for one species of bacteria and many are only able to lyse
specific strains within a species. This limited host range can be advantageous,
in principle, as phage therapy should result in less harm to the normal
body flora and ecology than commonly used antibiotics, which often disrupt the
normal gastrointestinal flora and result in opportunistic secondary infections
by organisms such as C. difficile [54]. The potential clinical disadvantages
associated with the narrow host range of most phage strains can be addressed
through the development of a large collection of well-characterized phage for a
broad range of pathogens, and methods to rapidly determine which of the phage
strains in the collection will be effective for any given infection.
Ideally, broad-host-range phage should be selected for therapeutic
applications. However, if such phage strains cannot be found, present molecular
techniques can be used to enhance the host range of some phage strains. For
example, it has been found that coliphage K1-5 is a ’dual’ specificity phage
that encodes two different tail proteins; this allows it to attack and
replicate in both K1 and K5 strains of E. coli [55]. One tail protein
found on phage K1-5 is a lyase protein, similar to that of phage K5 (specific
for the K5 polysaccharide capsule), and a second tail protein found on this
phage is an ENDOSIALIDASE similar to a tail protein found in phage K1E
(specific for the K1 polysaccharide capsule). In addition, the genomic region
encoding these proteins is almost identical to the genomic construct found in
the salmonella phage SP6, which codes for a protein that binds to the
salmonella O-antigen [56]. The observation of a similar tail genome motif in
both the salmonella phage SP6 and the coliphages K1E, K5 and K1-5 indicates
that this genomic construct might serve in the development of a modular phage
platform that could operate over a wide bacterial host range.
Other mechanisms have been found that permit expansion of the bacterial host
range of phage. These include the site-specific recombination systems that
permit phage to switch between alternative tail fibre proteins [57] and the use
of a reverse transcriptase, possessed by a Bordetella phage, to generate
variation in its tail fibre proteins [58]. Such expanded host-range ’platform’
phage would provide for versatility and save time and effort compared with that
required for the development of completely new phages for each bacterial
strain.
Other factors can affect host specificity. For example, bacterial
restriction/modification systems might limit the host range of some phage. This
problem could be addressed, in principle, by engineering phages with genomes
that do not contain restriction sites recognized by the non-permissive host.
Alternatively, phages could be produced in bacterial strains that provide DNA
modification(s) that allow the phage to escape restriction in the targeted
strain of bacteria. Another approach would be to incorporate genes into the
phage genome that facilitate inhibition of bacterial restriction/modification
enzymes, as exemplified by the mechanism used by the phage T7 which encodes an
antirestriction enzyme [59]. A construct containing such an antirestriction
gene might be adapted for use in other phage strains, or it might be possible
to modify T7 phage to expand its bacterial host range for E. coli
infections.
Phage growth: in vitro versus in vivo
In addition to the factors addressed above, bacteria grown with standard
laboratory protocols can behave differently in the milieu of an infection.
Bacteria possess feedback mechanisms that can alter their gene expression in
response to changing environmental conditions. Such variations in gene
expression can affect phage susceptibility. For example, Karakawa noted that S.
aureus rarely expresses the capsular polysaccharides found in clinical
isolates when the bacteria are grown in the laboratory [60]. Given the
possibility for such changes in the bacterial capsule, phage discovered using
bacteria grown in vitro might not be able to multiply in an infected
animal. Recently, it has been reported that phage that infected certain strains
of E. coli that did not express the cell surface protein Ag43 in
standard laboratory growth media can be inhibited by concentrations of bile
salts similar to that found in the gastrointestinal tract [61]. In this case,
the bile salts might affect the expression of the Ag43 protein, which has been
shown to be a phase -variable protein whose expression is associated with E.
coli BIOFILM formation [62].
In addition, different in vitro and in vivo bacterial
densities can be important [47]. Variations in phage physiology also are
important, as some phage infections cause the host bacteria to release lysins
that result in the destruction of bacteria not directly infected by the phage
([Box 1] and Fig. 4). In the early phage literature, there are reports of body
fluids (for example, serum, pus, ascites fluids, cerebrospinal fluid, urine and
bile) that inhibit the infectivity of phage that were active in vitro
against typhoid, colon bacilli and staphylococci [63, 64]. Some of these
effects might have resulted from alterations of bacterial physiology.
Detrimental phage genes
Some phage strains, the lytic phage, kill bacteria, whereas others --
LYSOGENIC or temperate phage -- have a dual life style: they can either kill
and lyse their bacterial host or become quiescent by integrating their genome
into their bacterial host chromosome. Some lysogenic phage encode toxins or
factors that enhance bacterial pathogenesis (Table 1). Phage can also
contribute, through transduction, to the transmission of antibiotic-resistance
genes [65]. It is essential that phage considered for therapeutic applications
be screened for toxin genes, either biochemically or by sequencing their DNA.
The presence of toxin, antibiotic resistance genes, and genes that increase
bacterial pathenogenicity, can be checked by searching phage genomes against
GenBank online using the Basic Local Alignment Search Tool (BLAST) [66] and
other similar programs. Success cannot be assumed as we are still in the
discovery stage of such detrimental genes. The usefulness of bioinformatic
analysis will increase as knowledge of these potential deleterious genes
accumulates.
Selection of therapeutic phage strains
The narrow host range of most phage strains dictates the need for the rapid
determination of bacterial susceptibility. The determination of suitable
therapeutic phage strain(s) using traditional procedures can take days to
accomplish, limiting the use of phage therapy to slowly progressing infections. However,
recent methods have been developed that permit the identification of both the
infectious agent and a suitable phage strain within a day or less.
One such approach uses of phage that contain reporter genes, such as luciferase
[67, 68]. For example, a collection of phage strains, each encoding luciferase
protein, could be individually placed in a multi-well plate. When a clinical
sample, such as urine or sputum, is added to the wells, light will be emitted
and detected in those wells that contain a phage strain that successfully
infects the bacteria in the clinical specimen. Such light emission would serve
to identify both bacterial strains and the phage strains that could be used
against them. This test could be performed in hours, instead of the days that
traditional culture methods require. This approach has been used to detect Listeria
contamination in foods [67] and in an inexpensive and rapid diagnostic test for
tuberculosis [68]. Alternatively, similar results can be achieved by using
phage that do not have such marker genes, by placing luciferin and luciferase
in the phage/bacterial incubation mixture in each of the wells of the
multi-well plate. The lysis of bacterial strains by a phage strain in any of
the wells will result in the discharge of adenylate kinase into the well, which
will convert the ADP in the reaction mix to ATP. As the luciferin/luciferase
system can utilize the ATP for light emission, lysis will serve to identify
phage susceptibility without the need to genetically engineer the phage with a
luciferase reporter gene [69].
Another method for the rapid identification of bacterial strains could be
provided by mass spectrometry (MS). Mass fragment ’fingerprints’ of lipid,
protein and nucleic acid bacterial components are used at present for rapid
strain identification [70, 71]. It might also be possible to use this approach
to determine whether bacteria are susceptible to a particular phage strain.
However, such information is not available at present and it might be
impractical to gain sufficient knowledge of bacterial mass fingerprints to
determine which phage strains to use therapeutically for a bacterial host
responsible for an infectious disease. Alternatively, phage gene products might
provide for the development of markers for both bacterial identification and as
an indicator of phage susceptibility. In this approach, one could use MS by
placing a clinical sample in growth media to amplify the infecting bacteria
followed by exposure to selected ’therapeutic’ phage strains. If the bacteria
were susceptible to the phage, MS would detect signature fragments of phage proteins
that are expressed only when infection of the bacteria occurs by a specific
phage. Such ’signature’ fragments, that are not part of the phage virion, would
be generated from phage: RNA polymerase, regulatory protein or lytic enzymes.
DNA microarray technologies also offer possibilities for determining
bacterial strains in disease states and possibly the phage strains that might
be used therapeutically. DNA microarrays, in conjunction with polymerase chain
reaction, are now being developed for the rapid diagnosis of bacterial strains
and antibiotic susceptibility [72]. In principle, it might be possible to
develop such methods for the determination of bacterial strains and their phage
susceptibility.
Development and preparation of phage
In addition to biological factors, phage preparative methods are crucial for
the development of reliable phage therapeutics. Early therapeutic applications
used impure phage preparations with deleterious clinical effects ([
In developing phage purification procedures, testing for adverse effects
should not be limited to observation with healthy animals. Individuals that are
under stress can have a lowered tolerance to endo- and exotoxins. In a recent
mouse bacteraemia study, a lower survival rate was observed in a ’control
experiment’ in which a phage strain, known to be inactive against the bacterial
strain being used at the LD50 level, was administered to the
infected mice. Although the highest doses of this phage preparation produced no
apparent adverse effects in healthy uninfected animals, an increased mortality
was observed in the bacteraemia-stressed mice. This increased mortality was
phage-dose dependent, indicating that stressed animals are more sensitive to
the phage itself, or to the trace amounts of endo- and exotoxins present in the
phage preparations, than normal animals [21]. This example provides additional
evidence for the need for highly purified phage preparations for therapeutic
applications.
It should also be noted that bacteriostatic or bacteriocidal agents, used to
ensure that no active bacteria are present in phage preparations, can also be
detrimental. The association between ’weak’ phage preparations and the presence
of organomercury compounds was made in a 1932 study of commercial phage
preparations from a large US pharmaceutical company [10].
Future prospects
Although results from animal experiments are encouraging, continuing
research will be needed to develop phage therapy for the treatment of human infectious
diseases. These efforts should include studies of phage genomics,
pharmacokinetics and efficacy in animal models of infectious diseases. In
addition, phage chosen for therapeutic applications will need to be screened to
reduce the chance that they carry genes encoding toxins, or factors that
enhance bacterial pathogenicity. Phage growth, purification and storage
protocols are needed to assure therapeutic efficacy and to reduce the
possibility of contamination of pharmaceutical preparations by toxins and
bacterial debris. Furthermore, the narrow host range of most phage strains
requires the development of rapid methods for the determination of appropriate
phage strains for use in specific infections.
Development of therapeutic phage could provide some relief from the growing
threat from the emerging antibiotic-resistant bacterial strains and, as
Lederberg suggested, treatments for epidemics such as cholera in refugee camps
[74]. In addition, the narrow host range of phage could be better suited than
presently employed antibiotics to a number of clinical applications. For
example, the lack of genetic variability in antibiotic-resistant bacteria
suggests that the resulting pathogenic bacteria might offer ideal targets for phage therapy.
Only 10 strains of Pneumococcus are associated with 75% of the cases of
antibiotic-resistant childhood pneumonia, and one-half of these cases are
caused by the single strain ’
Phage, with their narrow host range, could also prove useful in treating bacterial
infections in agricultural applications without disturbing larger ecological
systems, as is often the problem with antibiotics [74]. This suggestion is
strengthened by the recent observations that many antibiotic-resistant
bacterial strains are arising through clonal selection. In recognition of this
growing problem, the FDA recently announced that it is re-evaluating livestock
antibiotics, and it is now requiring manufacturers of proposed livestock
antibiotics to determine whether these proposed antibiotics will be associated
with the emergence of pathogenic organisms with resistance to drugs presently
in use for the treatment of human diseases [76].
In regard to concerns over regulatory agency approval, it should be noted
that phage have been used successfully as a means to probe immune-deficiency
diseases in human studies for the past three decades [49, 50]. In addition,
some vaccines were found, in the 1970s, to be contaminated with phage. An
executive order was issued to permit the continued use of these contaminated
vaccines [77, 78]. We should also recognize that we are normally in contact
with phage throughout our lifetime, with the complex interactions of bacteria
and phage in our colon, upper respiratory system and on our skin. In fact, many
present phage collections were derived from human waste.
It is clear from recent experiments that phage
therapy has the potential to rescue
animals infected with antibiotic-resistant bacterial stains [22, 21]. We now
also have much of the knowledge needed to develop phage into reliable
therapeutic preparations. Whether we embark on efforts needed to develop
therapeutic phage for human infections depends in part on our need to obtain
relief from the growing threat of emerging antibiotic-resistant bacterial strains
and our will to accomplish this task.
Definition List:
ADAPTIVE IMMUNE SYSTEM: The arm of the immune system that mounts an
antigen-specific immune response as the result of the clonal selection of
antigen-specific lymphocytes. Such lymphocytes produce antibodies that react
with the antigen. The adaptive immune responses differ from the innate and
non-adaptive immune system, which does not depend on clonal selection of
antigen-specific lymphocytes.
BIOFILM: A structure made up of a community of bacteria composed of
microcolonies and water channels that survives at a liquid interface. Such
biofilms play a role in the pathogenic effects of bacterial infections
associated with gingivitis, colitis, vaginitis, urethritis, conjunctivitis and
otitis.
ENDOTOXINS: Components of bacterial cells that are usually associated with
the lipopolysaccharide components of the outer layer of Gram-negative bacterial
cell walls that are toxic (to mammals). Endotoxins are released in large
quantities upon lysis of Gram-negative bacterial cells.
ENDOSIALIDASES: Enzymes that cleave at the sialic acid residue sites of the
complex oligosaccharides associated with the protective capsule of many
bacterial strains.
EXOTOXINS: A broad class of factors released by pathogenic bacteria that can
harm infected mammals. Examples of such exotoxins are botulism toxin (Clostridium
botulinum ), streptolysins (Streptococcus pyogenes ) and diphtheria
toxin (Corynebacterium diptheriae ).
IATROGENIC: An effect that is induced in a patient by a physician’s activity
or therapy; such effects often occur as complications of treatments for
infectious diseases.
LD50 : The amount of a substance that causes the death of 50% of
test subjects.
LYSATE: The colloidal bacterial growth media remaining after phage replicate
and kill the host cells. Lysates contain phage progeny, bacterial cell wall
debris and, often, internal cellular components (for example, proteins, nucleic
acids, small molecules and so on).
LYSOGENIC PHAGE: Phage that are capable of integrating their genome (that
is, lysogenize) into the host chromosome. Such phages often mediate horizontal
gene transfer (transduction) between bacterial strains. Most lysogenic phage
can also go through a lytic cycle to produce more phage, often after induction (from
some environmental factor).
LYTIC PHAGE: Phage that infect bacterial cells to replicate and then lyse
the bacterial host.
NEO-ANTIGEN: An antigen for which animals or humans being studied have no
pre-existing antibodies. The phage [phi]X174, which is highly immunogenic, has
served as such a neo-antigen in studies of human antibody responses, as most
humans have no pre-existing antibodies to this phage.
PHAGE: Bacterial viruses. The term phage is used as both singular and plural
when referring to phage(s) that is/are member(s) of a single phage strain.
However, when referring to phage in more than one strain the plural is phages.
PHAGE PLAQUE: The lesion formed when a phage particle is applied to a film
of a susceptible bacterial strain that is growing on an agar surface. The
lesion results from the infection of a bacterial cell by a phage particle,
followed by the production of phage progeny and their release by lysis,
followed by the infection and lysis of additional bacterial cells in the
vicinity of the initial infection.
Web Link(s):
DATABASES
Online Mendelian Inheritance in Man
Severe combined immunodeficiency:
http://www.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?102700
FURTHER INFORMATION
Encyclopedia of Life Sciences
Bacteriophages:
http://www.els.net/els/FDA/default.asp?id=25CA4A8E-4CC9-4DCE-9E33-D5DD1D5A2AE2
bacteriophages in industry:
http://www.els.net/els/FDA/default.asp?id=5CF03976-9CBE-4DCB-9A53-D4CBFA9BD344
phage display technologies: http://www.els.net/els/FDA/default.asp?id=CED7B675-3979-43EF-BB87-8EE22D7BE08C
Genomes of the T4-like Phages: http://phage.bioc.tulane.edu/
Phage Ecology and Evolutionary Biology:
http://www.mansfield.ohio-state.edu/~sabedon/
Phage Page: http://www.mbio.ncsu.edu/esm/phage/phage.html
Phage Therapy:
http://www.evergreen.edu/phage/phagetherapy.html
Therapeutic uses of phage: http://surfer.iitd.pan.wroc.pl/phages/phages.html
Box 1 | Therapeutic use of phage products
Phage gene products might also serve as therapeutic agents. Although such
applications lack the exponential growth capacity of phage, they could still be
highly effective. For example, the small-genome phages [phi]X174 and Q[beta]
encode polypeptides that could be developed into a new class of antibiotics, as
they interfere with bacterial cell wall biosynthesis [79]. Such inhibition
results in bacterial lysis. Similarly, phage-encoded endolysins that disrupt
the peptidoglycan matrix of the bacterial cell wall, and phage-encoded holins
that permeabilize bacterial membranes, can also serve as effective
antibacterial agents. A phage lysin, specific for streptococci groups A, C and
E, has been used to treat experimental upper respiratory infections in mice
[80]. Such lysins should be less disruptive than most antibiotic treatments as
they have little, if any, effect on other commensal organisms in the oral and
upper respiratory tract. In another example, the [gamma] phage of Bacillus
anthracis encodes a lysin that proved to be effective in rescuing mice
infected with Bacillus cereus , a bacterial strain closely related to B.
anthracis [69]. No resistant B. cereus strains were detected
following such treatment. In addition, phage lysin genes have been incorporated
into bacterial genomes for prophylactic applications. Gaeng et al . [81]
developed such a bacterial strain to secrete the functional phage lysin enzymes
Ply511 and Ply118 to reduce Listeria monocytogenes contamination in
dairy cheese production starter cultures [81]. These phage lysins can also be
used diagnostically. For example, when PlyG lysin destroys B. anthracis
, ATP is released, which, in conjunction with the luciferin/luciferase system,
results in the emission of light that can also be used to rapidly detect
bacilli and their germinating spores. This system was able to detect as few as
100 spores [69].
Box 2 | Therapeutic failure that might have resulted from impure phage
Some of the early clinical therapeutic failures might have been due to
inadequate purification of the phage preparations. In one such example,
reported in 1932, a phage strain was found that seemed to be promising as a
therapeutic agent as it could lyse cultures of plague (Yersinia pestis )
grown in broth media in less than 2 hours. However, when this phage strain,
purified solely by filtration, was injected into rabbits experimentally
infected with Y. pestis , the mortality increased to levels above those
found in infected rabbits that were not treated with phage. Furthermore, when
this phage preparation was used to treat 33 human patients, they all died. The
mortality from plague is normally 60-90% [82].
Caption(s):
Illustration 1: Timeline | Highlights in the development of phage as a
potential therapeutic agent for bacterial infections [see PDF for image]
Figure 1: Electron micrograph of phage. [see PDF for image]
This is an Escherichia coli phage. Phage can range from filamentous
to spherical structures. However, many of them have a distinct head, containing
their DNA, and a tail-like structure that can bind to bacterial receptors.
Figure 2: Systemic distribution of phage following intravenous and oral
administration of phage. [see PDF for image]
These experiments demonstrate that oral administration is not an effective
method for the delivery of phage to systemic sites as the blood and tissues
levels were seven to eight orders of magnitude lower with oral administration
than those achieved by systemic administration of phage. pfu, plaque-forming
units.
Figure 3: The effect of therapeutic phage concentration on morbidity and
mortality. [see PDF for image]
When phage are used to treat a systemic bacterial infection, the
concentration of phage administered must be adequate to kill the infecting
bacteria before they can kill the mammalian host. This point is illustrated in
the data, from a study using phage to treat bacteraemic mice infected with 109
colony-forming units (cfu) of vancomycin-resistant Enterococcus faecium
[21]. This concentration of bacteria normally results in death within 48 hours.
Each bar represents a single mouse, and all but the two control mice (injected
with buffer rather than bacteria, represented by the pink bars) were infected
with 109 cfu of bacteria. The phage concentrations administered 45
minutes after the bacterial infection are depicted in the key. The mice in the
last group (red bars) did not receive phage
therapy. The state of health scale is a
non-parametric scale in which: 5 = normal; 4 = decreased activity and ruffled
fur; 3 = lethargy, ruffled fur and hunchback posture; 2 = hunchback posture and
partially closed eyes with exudates; 1 = moribund; and 0 = death. pfu,
plaque-forming units.
Figure 4: Phage plaques on a bacterial ’lawn’. [see PDF for image]
The PHAGE PLAQUES illustrated on this plate display a mixture of
morphologies, which reflect the different phage strains applied to this
bacterial plate. Some of the phage produce small clear plaques, whereas other
phage strains that produce lysins have a clear centre zone surrounded by a
spreading zone of killing.
Table: Phages that carry toxin genes and their gene products [83] [see PDF
for image]
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Author Affiliation(s):
[1] Section on Biochemical Genetics, National Institute of Mental Health,
National Institutes of Health,
Email: merrilc@helix.nih.gov
Email: dscholl@helix.nih.gov
[2] Section of Developmental Genetics, National Cancer Institute, National
Institutes of Health,
Email: sadhya@helix.nih.gov
Author Bio(s):
Carl R. Merril is the chief of the Section on Biochemical Genetics at the
National Institute of Mental Health (NIHM), National Institutes of Health
(NIH). He has co-authored 225 articles, and 23 patents. His research ranges
from basic and applied bacteriophage studies to the development of sensitive
protein detection methods, including the silver stains. One of his publications
concerning this methodology was declared a citation classic by the journal Current
Contents as it earned more than 2,500 citations since its publication. In
addition, two of his patents in this field have earned recognition as top
money-makers for the Public Health Service (PHS), defined as a PHS-invented
commercialized product that exceeds US $100,000 in annual sales. He has
received a number of awards including the PHS Distinguished Service Medal and
the Surgeon General’s Exemplary Service Medal.
Dean Scholl is a fellow in the Section on Biochemical Genetics at the NIMH,
NIH. He has co-authored eight papers and three patents.
Sankar L. Adhya is the chief of the Section on Developmental Genetics,
National Cancer Institute, NIH. He has co-authored more than 150 articles and
10 patents. He has received a number of awards, including election to the US
National Academy of Sciences, the
DOI: 10.1038/nrd1111
