Download Phage therapy: Facts and fiction

ARTICLE IN PRESS
International Journal of Medical Microbiology 296 (2006) 5–14
www.elsevier.de/ijmm
REVIEW
Phage therapy: Facts and fiction
Mikael Skurnika,!, Eckhard Strauchb
a
Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, P.O. Box 21, FIN-00014,
and Helsinki University Central Hospital Laboratory Diagnostics, Helsinki, Finland
b
Federal Institute for Risk Assessment, Berlin, Germany
Received 15 April 2005; received in revised form 5 September 2005; accepted 12 September 2005
Abstract
Recent examples of the use of bacteriophages in controlling bacterial infections are presented, some of which show
therapeutic promise. The therapeutic use of bacteriophages, possibly in combination with antibiotics, may be a
valuable approach. However, it is also quite clear that the safe and controlled use of phage therapy will require detailed
information on the properties and behavior of specific phage–bacterium systems, both in vitro and especially in vivo.
In vivo susceptibility of bacterial pathogens to bacteriophages is still largely poorly understood and future research on
more phage–bacterium systems has to be undertaken to define the requirements for successful phage treatments.
r 2005 Elsevier GmbH. All rights reserved.
Keywords: Phage therapy; Yersinia enterocolitica; Escherichia coli
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . .
Principles of phage biology . . . . . . . . . . . . .
Bacteriophage receptors and phage resistance
Prerequisites for phage therapy . . . . . . . . . .
Phage (pharmaco)kinetics . . . . . . . . . . . . . .
Phages may carry harmful genes . . . . . . . . .
Phage products . . . . . . . . . . . . . . . . . . . . .
Recent in vivo phage studies . . . . . . . . . . . .
Phage therapy trials in chickens . . . . . . . . . .
Phage therapy in fish aquacultures . . . . . . . .
Phages riding inside a Trojan horse . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . .
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!Corresponding author. Tel.: +358 9 1912 6464; fax: +358 9 1912 6382.
E-mail address: [email protected] (M. Skurnik).
1438-4221/$ - see front matter r 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ijmm.2005.09.002
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ARTICLE IN PRESS
6
M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14
Introduction
Principles of phage biology
In the April 2003 issue of ‘‘New Scientist’’, James
Randerson wrote an article ‘‘Virus cleans up food poisoning
bug’’ (Randerson, 2003) where he reported observations
presented by Andrew Brabban (Evergreen State College in
Washington state) in the XXth Annual Meeting of the
Society for General Microbiology in Edinburgh, UK.
Brabban had wanted to test the effect of different
antibiotics on Escherichia coli O157:H7 in infected sheep.
However, the researchers faced an unexpected problem; the
bacteria disappeared from the infected sheep very rapidly. It
turned out that the sheep carried a bacteriophage specific
for E. coli O157:H7 and the phage efficiently eliminated all
the inoculated pathogens from the sheep tissues. This is an
example of a number of observations made on the
antibacterial potency of bacteriophages during the last 90
years after the independent discoveries of bacteriophages by
d’Herelle and Thwort.
The prospect of phage therapy has stimulated a lot of
discussion recently. The increase in interest can be
explained in part by the publication of experiments
conducted using phage lysins (Loeffler et al., 2001;
Nelson et al., 2001; Schuch et al., 2002) and by animal
experiments where the bacterial infections are challenged with live bacteriophage particles (Biswas et al.,
2002; Broxmeyer et al., 2002; Cerveny et al., 2002;
Loeffler et al., 2003; Merril et al., 1996; Sulakvelidze
et al., 2001; Westwater et al., 2003).
Steve Projan (2004) presented a number of provocative arguments recently challenging the optimism
regarding phage therapy. Apparently, Projan dislikes
proponents of phage therapy since he puts forward such
expressions like ‘‘a cult of phage therapy followers’’,
‘‘the little animal efficacy data there is in the literature
can charitably be described as meager’’, ‘‘this silence (on
animal efficiency data) speaks volumes’’ and ‘‘anecdotal
testimonials of former patients’’.
We will not try to cover old literature in this review
since there are excellent recent reviews on phage therapy
available (Alisky et al., 1998; Anonymous, 1983; Bradbury, 2004; Bull et al., 2002; Cerveny et al., 2002; Dixon,
2004; Duckworth and Gulig, 2002; Inal, 2003; Levin and
Bull, 2004; Merril et al., 2003; Nakai and Park, 2002;
Payne et al., 2000; Pirisi, 2000; Projan, 2004; Schoolnik
et al., 2004; Stone, 2002a, b; Sulakvelidze et al., 2001;
Summers, 2001; Thacker, 2003; Thiel, 2004; WeberDabrowska et al., 2002, 2003; Weinberg, 2002). We will
focus mainly on recent work carried out to exploit the
therapeutic potential of phages and phage products to
combat bacterial pathogens. Another interesting aspect
of phage application is the use of whole phage particles
to deliver vaccines in the form of immunogenic peptides
attached to modified phage coat proteins or as delivery
vehicles for DNA vaccines, which was recently reviewed
by Clark and March (2004).
Bacterial viruses (bacteriophages) occupy all those
habitats of the world where bacteria thrive. It has been
estimated that for each bacterial cell there are ten
bacteriophage particles. Recently, the existence of
viruses specific for archaebacteria (archeophages) has
become evident also. Some phages are highly specific
while others are extremely broad in their host range.
Bacteriophage taxonomy is based on their shape and
size as well as on their nucleic acid. Most bacteriophages
have ds DNA, however, some have ss DNA, ds RNA or
ss RNA.
Upon infection of the bacterial host different phages
can have quite different fates. Some phages follow the
lytic infection cycle whereby they multiply in the
bacterial cell and lyse the bacterial cell at the end of
the cycle to release newly formed phage particles. Some
phages may use the lysogenic pathway where the phage
genome will integrate as part of the host genome,
replicate as part of the host genome and stay in a
dormant state as a prophage for extended periods of
time. If the host bacterium encounters adverse environmental conditions the prophage may become activated
and turn on the lytic cycle, at the end of which the newly
formed phage particles will lyse the host cell.
The following phases can be distinguished in the lytic
bacteriophage developmental cycle:
1. Adsorption of the phage on the bacterial cell by
binding to a specific receptor.
2. Injection of the nucleic acid into the bacterium.
3. Expression of the phage early genes, synthesis of
early proteins, most involved in the shutting down of
the host bacterium systems and phage genome
replication.
4. Replication of the phage genome.
5. Expression of the phage late proteins involved in the
formation of new phage particles and lysis of the host
bacterium.
6. Assembly of the phage heads and tails and packaging
of the genome.
7. Lysis of the host bacterium and release of the new
phage progeny.
The ability of the phages to kill the bacterial cells at
the end of the infectious cycle is the cornerstone of the
idea of using phages as therapeutic agents. However, for
a positive outcome of the therapeutic use of bacteriophages all the above listed steps in the phage infectious
cycle need to take place. Bacteriophages have coevolved
with their bacterial hosts. Already in the few thoroughly
studied phage–bacterium systems many examples of
intricate molecular mechanisms have been revealed.
Therefore, one cannot expect that all the phage/host
systems will behave identically under the conditions met
ARTICLE IN PRESS
M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14
in vivo; on the contrary, it would be surprising to find
two identically behaving systems.
Bacteriophage receptors and phage resistance
One essential step in phage infection is the attachment
of the phage onto the bacterium. For this purpose
phages can use bacterial capsules, different parts of
lipopolysaccharide (LPS), flagella, fimbriae and many
other surface proteins as receptors. Bacteriophages may
also use enzymes to break down capsule-like materials
on the bacterial surface in a drill-like manner to reach
the cell wall of the bacterium. A good example of such a
phage is PK1A that uses the endosialidase activity of its
tail to degrade the polysialic acid capsule of E. coli K1
(Pelkonen et al., 1992).
By mutating or losing the phage receptor bacteria
become resistant to phages in question. This may not
always be bad (Levin and Bull, 2004) since resistance
may reduce the fitness of the bacteria or if the receptor
used by the phage is a virulence determinant, loss of the
receptor would decrease the virulence of the bacterium
dramatically.
A bacterium may become phage-resistant also by
lysogeny (see above), whereby the bacterium becomes
immune to the phage and to its close relatives, or by
acquiring horizontally a restriction-modification system
that degrades the injected phage nucleic acid. In
addition, phage resistance may be caused by a mutation
in a gene the product of which is essential for the phage
replication or assembly.
Prerequisites for phage therapy
Levin and Bull (2004) suggest that phage therapy only
needs to decrease the numbers of infecting bacteria to a
level where the host defenses can take care of the
remaining bacteria. They also write that it is difficult to
understand why a phage that replicates extremely well in
the target bacteria fails when it is used for treatment.
Understanding this requires a quantitative appreciation
of the dynamics of the phage infection process,
especially in vivo.
Before attempting phage therapy several, sometimes
rather demanding, prerequisites should be met:
1. Phage therapy should not be attempted before the
biology of the therapeutic phage is well understood.
Since the phage–host systems are extremely complicated, this prerequisite has to be faced with some
common sense.
2. Phage preparations should meet all the safety
requirements; the preparations should be free of
bacteria and their components.
7
3. Phage preparations should contain infective phage
particles, thus storage of the preparations should be
validated.
4. The phage receptor should be known. In a bacterial
population of 106–108 bacteria there is a high
possibility of spontaneous phage-resistant mutants
deficient in the receptor or with an altered receptor. It
can be assumed that a mutation eliminating the
receptor that functions as a virulence factor of a
pathogen (such as LPS) would attenuate the bacterium and then it would be easier for the host immune
system to eliminate the bacteria.
5. The efficacy of phage therapy should be tested in an
animal model. Each phage may behave differently in
vivo.
Before a phage as a therapeutic agent can reach the
end user many bureaucratic and regulatory hurdles need
to be cleared even when the above prerequisites are
properly met.
Phage (pharmaco)kinetics
Surprisingly little detailed research has been performed on the fate of phages in animals and humans.
The pharmacokinetics are complicated due to the selfreplicating nature of phage. The in vitro growth data for
a phage cannot be directly applied to the in vivo
situation, in addition the in vivo data for one phage
cannot be transferred to another phage. The use of
phages as drugs may differ dramatically from pharmaceuticals/antibiotics due to differences in the phage
pharmacokinetics (Payne and Jansen, 2003). Critical
parameters that affect phage therapy are the phage
adsorption rate, burst size, latent period and initial
phage dose, also density-dependent thresholds and
associated critical times should be considered (Payne
and Jansen, 2001). Another critical parameter is the
clearance rate of the phage particles from the body fluids
by the reticuloendothelial system.
Payne has developed mathematical models to predict
the behavior of phages in vivo taking into account
population dynamics (Payne and Jansen, 2003). Two
paradoxical observations should be mentioned here. It is
certain that timing of the phage treatment is critical and
that phage administered too early may result in clearing
of the phage from the body before it reaches the
replication threshold. Similarly, addition of antibiotics
in parallel with phage may hamper the phage efficacy.
The replication threshold and proliferation threshold
are expressions used to describe the situation where a
low concentration of host bacteria is present in the
culture and it takes some time before the bacteria reach
a density where a net increase in phage concentrations
ARTICLE IN PRESS
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M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14
can be seen (Kasman et al., 2002). Apparently, the
threshold depends on the rare encounters of the phage
with the relatively few host cells. In vivo experiments
should demonstrate also whether the physical and
chemical conditions encountered in vivo support the
phage life cycle. In some body compartments bacteria
may reside in an environment that does not provide
phages with optimal conditions for infection.
Weld et al. (2004) studied mathematical models of
phage growth and also tried to fit the formulae to
experimental data. They used T4 and K1-5 phages and
E. coli strains as host bacteria and rats as the animal
model. They concluded that their mathematical model
failed to predict phage growth in the rats demonstrating
the complexity of phage growth in vivo. Coexistence of
the host bacterium and its specific phage was studied by
Fischer et al. (2004). They showed that within a bacterial
population a fraction of cells may exist in a phageresistant form, thus indicating heterogeneity within the
population.
Bull et al. (2002) presented an analysis of the now
classical Smith and Huggins phage therapy experiments
(Smith and Huggins, 1982, 1983; Smith et al., 1987) and
repeated some of their experiments. They also introduced and used a resistance competition assay (RCA)
and a phage replication assay (PRA) to analyze the
parameters involved in the phage treatment.
Phages may carry harmful genes
A full knowledge of phage genome sequences would
be helpful to evaluate possible complications during
phage therapy. For example, phages may carry virulence
factor or toxin genes (Brüssow et al., 2004; McGrath et
al., 2004). Mesyanzhinov et al. (2002) determined the
complete nucleotide sequence of Pseudomonas aeruginosa-specific phage fKZ, the largest bacteriophage sequenced thus far (ca. 280 kb with 306 predicted genes),
and observed that a number of fKZ gene products has a
strong similarity to functionally unknown proteins from
a diversity of organisms. This observation must be taken
into consideration before the therapeutic use of phages
(sequenced or not sequenced) is attempted because some
gene products might be toxic for humans.
The in vivo phage-associated conversion of Tox!
Streptococcus pyogenes into Tox+ bacteria (Broudy and
Fischetti, 2003) is an example of the phage-related
dangers encountered in nature and it can serve as a
warning of the possible side effects of bacteriophage
therapy. Included in this category are other phageassociated toxins, of which the CTX cholera toxin (Boyd
et al., 2000; Davis et al., 2000; Davis and Waldor, 2003),
botulinum toxin (Brüssow et al., 2004; Eklund et al.,
1971), shiga-toxin (Beutin et al., 1999; Strauch et al.,
2001b, 2004), and diphtheria toxin (Brüssow et al., 2004)
are the best known.
Phage products
Different types of purified bacteriophage products
have been evaluated as anti-infective agents. These
include bacteriophage lysins and bacteriophage tail-like
bacteriocins. One should keep in mind that lysins
usually work efficiently only for Gram-positive bacteria.
Bacillus anthracis is killed by the g phage lysin PlyG
‘from without’ and PlyG can protect mice infected by B.
anthracis (Schuch et al., 2002). Analogously, mureindegrading enzymes (lysozymes, amidases or endopeptidases) of phages infecting Streptococcus pneumoniae
have been used successfully in mouse infections (Jado et
al., 2003; Loeffler et al., 2001, 2003). A combination of a
lysozyme and amidase was found to have a synergistic
effect in vitro (Loeffler and Fischetti, 2003) and also in
protecting mice (Jado et al., 2003). Additional studies
revealed that combinations of a murein-degrading
enzyme with conventional antibiotics showed synergistic
activities in vitro. However, this has to be confirmed by
in vivo studies (Djurkovic et al., 2005). In a mouse
model, a single dose of a lysin significantly reduced
bacterial colonization by group B streptococci (S.
agalactiae) in both the vagina and oropharynx (Cheng
et al., 2005).
Phage tail-like bacteriocins is another group of phage
products able to eliminate target bacteria ‘from without’. This special group of bacteriocins consists of highmolecular-weight particles composed of fragments of
bacteriophages and they are produced by a number of
Enterobacteriaceae and other Gram-negative bacteria
(Bradley, 1967; Daw and Falkiner, 1996). Recently, a
phage tail-like bacteriocin, enterocoliticin, was isolated
from the culture supernatant of the Yersinia enterocolitica strain 29930. Upon contact with susceptible
Yersinia strains the phage tails contract and kill the
bacterial cell by forming pores in the cell wall leading to
a rapid loss of ions (Strauch et al., 2001a). The cell wall
receptor of enterocoliticin was identified as the outer
core hexasaccharide of the LPS present in certain
serotypes of Y. enterocolitica including serotype O:3
(Skurnik et al., 1995, 1999; Strauch et al., 2003). The
efficacy of enterocoliticin as an antimicrobial agent
against infections with pathogenic Y. enterocolitica O:3
strains was assessed recently (Damasko et al., 2005). In
cell cultures exposed to Y. enterocolitica enterocoliticin
killed bacteria adhering to the surface of eukaryotic cells
whereas intracellularly located bacteria were not accessible to the bacteriocin. In mice infected orally, an
increase in the Y. enterocolitica numbers was inhibited at
time points shortly after the oral application of
ARTICLE IN PRESS
M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14
enterocoliticin indicating that the particles were effective
on recently introduced pathogens. Repeated application
of enterocoliticin, however, did not prevent the colonization of the gastrointestinal tract by Y. enterocolitica
suggesting that the bacteria rapidly escaped out of the
reach of the enterocoliticin in vivo. On the other hand,
enterocoliticin survived well in the hostile environment
of stomach juices. Use of phage tail-like bacteriocins as
antimicrobial agents most likely will be limited only to
in vitro applications.
In this section could also be discussed the work of
Hagens et al. (2004) who describe the use of nonreplicating genetically modified phage Pf3R to kill P.
aeruginosa in a mouse model. They deprived the
filamentous phage Pf3 genome of the export protein
gene and replaced it with the restriction endonuclease
BglIIR gene. In this way the phage infects the host
bacteria expresses the restriction endonuclease BglII
that breaks the host genomic DNA but not that of the
phage (which is missing the recognition site) and kills the
host. In vitro Pf3R released only small amounts of
endotoxin from the bacteria when compared to a lytic
phage. In vivo experiments with infected mice also
demonstrated a protective effect against 3–5 times the
median lethal dose of bacteria (Hagens et al., 2004).
Recent in vivo phage studies
In a recent paper from Brüssow and colleagues
(Chibani-Chennoufi et al., 2004) systematical experiments with E. coli phages were conducted. A collection
of E. coli-specific phages were isolated from stool
samples of children in Bangladesh, environmental water
in Bangladesh and sewage in Switzerland. These phages
were then tested against a collection of E. coli strains,
both pathogenic (EPEC and EHEC) and non-pathogenic, the latter representing the ECOR collection
(Ochman and Selander, 1984). Altogether four phages
related to the well characterized T4 coliphage that
showed the widest and complementary infection range
among the strains were selected for the mouse experiments.
The endogenous E. coli gut flora of ten conventional
mice was assayed for phage susceptibility. Altogether
500 lactose-positive colonies were tested. The results
indicated that practically all E. coli cells were lysed by at
least one of the phages. The susceptibility of the E. coli
to the phages was then determined in vivo by applying
orally increasing doses of phages mixed in the drinking
water. At different time points, lactose-positive colonies
were counted from the stools, however, only a slight
decrease in the numbers was seen (counts dropped from
106.2 to 105.7). The recovered colonies were sensitive to
phage lysis and the phages survived the passage through
9
the gut although they did not multiply significantly.
Thus the survival of the bacteria in the gut during the
phage passage could only be explained by some
physiological reasons that prevented phage-induced
lysis. The authors went further and used axenic mice
that were infected with a single E. coli strain and then
were given phages in the drinking water. In these mice
the phage titers in the stools increased in one day from
the 105/ml in the drinking water to 1010/ml in the stool,
at the same time numbers of E. coli in the stools dropped
from 108 to 104, from where the numbers leveled off
during the subsequent days to 105. Again the recovered
colonies were sensitive to the phages suggesting that
they have resided in gut sites protected from phage.
Paisano et al. (2004) infected human dental roots with
Enterococcus faecalis to study the antimicrobial effect of
bacteriophages. They could show reduction of viability
of the bacteria in root canals when different phage:bacteria ratios were used.
In a very thorough study, Biswas et al. (2002)
performed experiments using a mouse model of vancomycin-resistant Enterococcus faecium (VRE) infection.
They isolated VRE-specific phages from raw sewage and
selected one that showed an antibacterial effect against
79% of the studied strains in vitro for animal experiments. They showed first that intraperitoneally (i.p.)
45 min post infection administered phage was able to
rescue mice from VRE bacteremia and that the rescue
was associated with a significant decrease in bacterial
numbers in blood. They also demonstrated that phage
administration before 5 h post infection still fully
rescued the mice while treatment delayed beyond 5 h
rescued only some of the mice (Biswas et al., 2002).
Bacteriophage fMR11 was tested in mouse infections
caused by Staphylococcus aureus (Matsuzaki et al.,
2003). The bacteriophage was isolated after induction of
S. aureus strains by mitomycin. It was selected for
experimental studies due to its broad ability to infect
and lyse the 75 S. aureus indicator strains. The 43-kb
phage genome was completely sequenced and did not
carry any known toxin, virulence factor or antibiotic
resistance genes. The authors also characterized the
phage with regard to its biological features. Mice were
infected i.p. with an S. aureus dose that killed 80% of
the untreated mice within 24 h and all within 7 d. Most
of the mice died between 6 and 7 h after infection. Mice
receiving phage i.p. immediately following the bacterial
challenge were protected. In an experimental design
where mice were infected with the fMR11 lysogen strain
no protection was observed allowing the authors to
conclude that a direct bactericidal effect of the phage
was the principal determinant of the protective effect
and not any indirect effect such as a phage-stimulated
immune response (e.g. production of cytokines)
(Matsuzaki et al., 2003). Phage could rescue mice when
administered 60 min later than the bacteria. Phage and
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bacterial numbers in the circulation were determined
after the infection and showed that the bacterial load
was much lower in the blood of phage-treated mice
when compared to those that received only bacteria.
They also noticed that phage titers in mice infected with
bacteria remained higher than the titers in mice that
received only the phage. This suggested that the phage
replicated in the infected mice and consumed the
bacteria. This happened until the bacteria disappeared
from the circulation which was paralleled by elimination
of the phage (Matsuzaki et al., 2003).
As an example of the failure in phage therapy we will
briefly describe our own phage therapy experiments that
were performed with Y. enterocolitica O:3 strains.
Strains of this serotype are low-pathogenicity strains
of Y. enterocolitica and cause only a mild diarrhea in
mice. Phage treatments were designed to test the
possibility of eradicating selectively pathogenic bacteria
from the gut microflora in healthy mice. Such an
application might be attractive to combat Y. enterocolitica O:3 in pigs which is the main source of human
infections. Y. enterocolitica serotype O:3 strains 6471/76
(Skurnik, 1984) and 13169 (Strauch et al., 2001a) were
selected for phage challenge studies. Eight-week-old
female BALBc mice were used in the experiments,
phages and strains were applied orally with a feeding
syringe in a volume of 0.5 ml per mouse.
Yersinia phage PY100 was isolated from pig manure
collected from a farm in Germany and chosen for the
experiments, as it formed in vitro large clear plaques
at 37 1C on cultures of Y. enterocolitica O:3. Phages
were administered orally in a single application with
a dose of 4 " 1010 pfu to study the dissemination of
phage particles in the mice. Five mice were killed after 6,
12, 24, and 48 h, and PY100 titres (pfu/g) were
determined in the following samples: ileum, caecum,
faeces, spleen, liver, and kidney. It was found that the
phage particles were active for at least 24 h within the
gastrointestinal tract (titres between 104 and 106 pfu/g
of organ and faeces, respectively). In some mice, phage
particles were even found in deeper organs indicating
that particles had passed the gastrointestinal barrier.
Since a single application of PY100 did not inhibit
the colonization of mice by Y. enterocolitica O:3 strain
13169, the experiment was repeated by administering
the phage at 24 h intervals four times starting 1 h after
the infection (Fig. 1; Kaspar, 2003). Mice were killed
after 24, 48, 72, 96, and 170 h (five mice per time
point). In Fig. 1 the recovered Yersinia cfu are shown:
mice killed after 24 h had received one phage dose,
mice sacrificed after 72 h had received three phage
doses and mice sacrificed after 170 h four doses. There
was no significant difference between mice treated
with PY100 phage and untreated mice. Yersinia titres
were in the range from 106 to 108 cfu/g of tissue or
faeces.
Fig. 1. Recovery of Y. enterocolitica O:3 13169 bacteria in
organs and faeces of mice after repeated application of phage
PY100. Each animal was infected with 1.8 " 109 cfu of strain
13169; each phage dose contained 1.85 " 1010 pfu per animal.
(!) control: only Yersinia infection; (+) mice treated with
phages. Bars represent mean cfu (5 animals per group),
standard deviation is indicated.
Another experiment on a smaller scale was carried out
with yersiniophage fYeO3–12 (Pajunen et al., 2000,
2001) as a protective phage. After oral administration
phage fYeO3–12 was also active in the gastrointestinal
tract for more than 24 h. Mice were infected with Y.
enterocolitica strain 6471/76. Each mouse was infected
orally with 1.9 " 109 cfu. In the control group, mice were
infected with Y. enterocolitica only. In the therapy group
all mice received phage (a dose of 5.5 " 108 pfu of
fYeO3–12) 1 h after the bacterial challenge and additional doses after 24, 48 and 72 h. After 6, 24, 48, 72, 96,
and 120 h two mice in each group were killed and
investigated for Y. enterocolitica O:3 (cfu/g) and
fYeO3–12 (pfu/g, only for therapy group). The results
obtained with phage fYeO3–12 were similar to those of
PY100, as untreated and treated animals yielded
approximately the same Yersinia titres in the gastrointestinal tract (titres between 105 and 108 cfu, Strauch
and Skurnik, unpublished data).
The major finding of our experiments was that phage
treatment was not able to eradicate the bacteria from the
mice even though in in vitro cultures the bacteria were
efficiently killed. In summary, these phage therapy trials
with Y. enterocolitica O:3 demonstrated that Y. enterocolitica colonization in mice cannot be controlled by
phage therapy, at least with the regimen used by
ourselves. It would not be surprising if similar results
are obtained with other bacterial pathogens.
Phage therapy trials in chickens
Huff and colleagues have explored the possibilities of
phage therapy using chicken challenged with a respiratory
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M. Skurnik, E. Strauch / International Journal of Medical Microbiology 296 (2006) 5–14
infection-causing Escherichia coli. Their first paper
reported just the clinical signs of the infected birds,
without analysis of the phage or bacterial loads (Huff
et al., 2002a, b). Phage mixed with E. coli prior to an air
sac infection protected chicken but phage administered
into the drinking water did not. In their second study they
used an aerosol spray of phage instead of phage in the
drinking water and concluded after observing some
protection against the infection that an aerosol spray
was a promising way of administration of the phage (Huff
et al., 2002a, b). In their third study an aerosol spray and
intramuscular injection of bacteriophage were compared
(Huff et al., 2003b). Phage given as an aerosol spray
immediately after the E. coli infection but not after 24 h
was protective, whilst the intramuscular phage protected
also when given immediately, 24 or 48 h after challenge.
Presence of phage in the blood was followed. Almost no
birds had phage in the blood after aerosol spraying whilst
high numbers were present after intramuscular administration (Huff et al., 2003b). Multiple intramuscular
treatments proved to be more effective than a single dose
(Huff et al., 2003a). Another clinical trial was conducted
to evaluate a combination of the antibiotic enrofloxacin
and intramuscularly administered bacteriophage (Huff et
al., 2004). Both treatments individually provided effective
treatments of the E. coli infection, but the synergy
between the two treatments led to a total protection of
the birds, thus suggesting a significant value of the
combined treatment.
Phage therapy in fish aquacultures
A Japanese group has studied the potential to use
phage to prevent fish infections caused by Lactococcus
garviae or Pseudomonas plecoglossicida (Park and
Nakai, 2003; Park et al., 2000). Phages were administered either i.p. or orally as phage-impregnated feed.
The authors also presented the idea of introducing the
phage inside bacteria to fish, and found no difference in
protective efficacy between them.
Phages riding inside a Trojan horse
The escape of invasive pathogens into closed tissue
and organ compartments may block the effective use of
bacteriophages especially if the phage cannot actively
follow the bacteria. Replicating phages may enter the
compartment inside an invading bacterium analogous to
the Trojan horse. It is however quite clear that many
variations of this theme will be encountered among
different bacteriophage/pathogen systems. In an attempt
to utilize the Trojan horse concept, Broxmeyer et al.
(2002) developed a system where Mycobacterium smeg-
11
matis, an avirulent mycobacterium, was used to deliver
the lytic phage TM4 into macrophages in tissue cultures
where both M. avium and M. tuberculosis resided. The
results showed that the treatment was able to reduce the
numbers of viable intracellular bacilli (Broxmeyer et al.,
2002).
Conclusions
Based on the few examples presented here, the use of
bacteriophages to control bacterial infections shows
therapeutic promise. The worldwide increase of pathogenic bacteria resistant to antibiotics makes it an
imperative to exploit alternative strategies to combat
this threat. The therapeutic use of bacteriophages –
perhaps in combination with antibiotics – may turn out
to a valuable approach. However, it is also quite clear
that the safe and controlled use of phage therapy will
require detailed information on the properties and
behavior of the specific phage–bacterium system, both
in vitro and especially in vivo. The in vivo susceptibility
of bacterial pathogens to bacteriophages is still largely
poorly understood.
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