ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2007, p. 1934–1938
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 51, No. 6
Phage Therapy of Pseudomonas aeruginosa Infection in a Mouse Burn
Catherine S. McVay,† Marisela Vela ´squez,‡ and Joe A. Fralick*
Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, Texas
Received 16 August 2006/Returned for modification 21 November 2006/Accepted 17 March 2007
Mice compromised by a burn wound injury and subjected to a fatal infection with Pseudomonas aeruginosa
were administered a single dose of a Pseudomonas aeruginosa phage cocktail consisting of three different P.
aeruginosa phages by three different routes: the intramuscular (i.m.), subcutaneous (s.c.), or intraperitoneal
(i.p.) route. The results of these studies indicated that a single dose of the P. aeruginosa phage cocktail could
significantly decrease the mortality of thermally injured, P. aeruginosa-infected mice (from 6% survival without
treatment to 22 to 87% survival with treatment) and that the route of administration was particularly
important to the efficacy of the treatment, with the i.p. route providing the most significant (87%) protection.
The pharmacokinetics of phage delivery to the blood, spleen, and liver suggested that the phages administered
by the i.p. route were delivered at a higher dose, were delivered earlier, and were delivered for a more sustained
period of time than the phages administered by the i.m. or s.c. route, which may explain the differences in the
efficacies of these three different routes of administration.
Pseudomonas aeruginosa plays a prominent role as an etio-
logical agent of serious infections in patients with burn
wounds. Acute burn wounds cause a breach in the protective
skin barrier and suppress the immune system, rendering the
patients highly susceptible to bacterial infection. P. aeruginosa
colonization of severe burn wounds and its rapid proliferation
within the damaged tissues often lead to disseminated infec-
tions, resulting in bacteremia and septic shock (8, 20) and high
rates of mortality and morbidity. Treatment of such infections
is confounded by the innate and acquired resistance of P.
aeruginosa to many antimicrobials (8, 15). It has been esti-
mated that at least 50% of all deaths caused by burns are the
result of infection (8), and untreatable infections have become
a tragically frequent occurrence in patients infected with P.
aeruginosa (9). Hence, the development of new therapeutic
and prophylactic strategies for the control of bacterial infec-
tion in patients with burn wounds is needed.
An alternative or supplement to antibiotic therapy, which is
currently being reexamined, is the use of bacterial viruses
(phage/bacteriophage) to target bacterial infections, i.e., phage
therapy (13, 16–18, 22, 29, 30–32). Soothill examined the ability
of bacteriophage to prevent the rejection of skin grafts of
experimentally infected guinea pigs (27). His findings demon-
strated that the phage-treated grafts were protected in six of
seven cases, while untreated grafts failed uniformly, suggesting
that phage might be useful for the prevention of P. aeruginosa
infections in patients with burn wounds. However, while mul-
tiple studies have demonstrated the benefits of phage therapy
for a variety of bacterial infections in animal model systems
(3–7, 10, 14, 19, 23–26, 33–35), little documentation exists with
regard to the treatment of burn wound infections (2). In the
study described here we used the mouse model of thermal
injury (28) to examine the efficacy of phage therapy in abro-
gating fatal P. aeruginosa infections. These studies include the
examination of different routes of phage administration.
(This work comprised part of Marisela Velasquez’s require-
ments for the master of science degree.)
MATERIALS AND METHODS
Bacterial strains, bacteriophages, and culture conditions. PAO1Rifis a
rifampin-resistant derivative of virulent P. aeruginosa strain PAO1 (12), which
was kindly provided by Abdul Hamood (11) and which was grown in Luria-
Bertani (LB) medium supplemented with rifampin (80 ?g/ml), 1 mM MgSO4,
and 1 mM CaCl2in a gyratory shaker at 250 rpm at 37°C.
The P. aeruginosa phages were plaque-purified subcultures of phages that had
been purchased from the American Type Culture Collection (Catalogue of
bacteria and bacteriophages, 18th ed., 1992; ATCC, Manassas, VA). Monophage
preparations were propagated on their respective hosts growing in LB medium at
37°C in a gyratory shaking water bath at 250 rpm. Phage lysates were centrifuged
(5,000 ? g for 15 min) to remove cellular debris, filter sterilized (pore size, 0.22
?m; Millipore), and stored over a drop of chloroform at 4°C in amber bottles.
The phage preparations to be used therapeutically were passed through a column
containing Detoxi-endotoxin removing gel (Pierce, Rockford, IL), as recom-
mended by the manufacturer, and eluted with pyrogen-free water. The eluted
phages were diluted to the appropriate titer with filter-sterilized phosphate-
buffered saline (PBS; pH 7.2) prior to administration to the mice. Phage titers
were determined by serial dilution and plaque assays by the soft overlay tech-
nique (1). This entailed the addition of 100 ?l of the different phage dilutions to
100 ?l of an overnight culture of their host strain. The mixture was then allowed
to stand at room temperature for 5 min for phage adsorption, after which 3 ml
of soft agar (0.7% LB agar maintained at 48°C) was added and the mixture was
poured over an LB agar plate. The soft agar overlay was allowed to solidify, the
plates were incubated overnight at 37°C, and the plaques were counted to
determine the phage titer.
Selection of therapeutic phages. To select for the phages to be used in our
phage cocktail, we used two criteria: virulence and host range (i.e., utilization of
different phage receptors). Of our 13 ATCC phages, 7 were able to grow on
PAO1Rif. Of those, two formed hazy plaques, suggesting that they may be
lysogenic. To determine the relative virulence of the remaining five phages, we
developed an in vitro virulence test in which we determined the MIC required to
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Texas Tech University Health Sciences Cen-
ter, 3601 4th Street, Lubbock, TX 79430. Phone: (806) 743-2555. Fax:
(806) 743-2334. E-mail: firstname.lastname@example.org.
† Present address: Biological Sciences Department, Auburn Univer-
sity, Auburn AL 36849.
‡ Present address: Kilimanjaro Christian Medical College, Tumaini
University, P.O. Box 2240, Moshi, Tanzania, Africa.
?Published ahead of print on 26 March 2007.
clear (lyse) an exponentially growing culture of PAO1Rif(?107/ml) over a given
period of time (5 h). Those with the lowest MICs were considered the most
virulent. We realize, however, that we are measuring only one virulence param-
eter and that in vivo and in vitro virulence may not be equivalent and may be
determined by other phenotypic traits (6, 21).
Resistance to phage often arises through bacterial mutations that alter recep-
tors on the bacterial surface to which the phage binds (phage receptors). In an
attempt to decrease the likelihood of the emergence of phage-resistant PAO1Rif
strains, we selected phages that utilized different phage receptors for infection.
To identify such phages we isolated PAO1Rifmutants resistant to each of the five
different phages being analyzed and determined the sensitivities of these mutants
to the other phages being analyzed. We assumed that a phage to which a
PAO1Rifphage-resistant mutant was sensitive did not share a common receptor
with the phage to which the mutant was resistant.
Based on our virulence and host range (receptor usage) tests, we selected
three phages for use in this study. The phage cocktails used in this study con-
tained approximately 108PFU/100 ?l inoculum of each of the following phages:
Pa1 (ATCC 12175-B1); Pa2 (ATCC 14203-B1), and Pa11 (ATCC 14205-B1)
(ATCC catalogue of bacteria and bacteriophages, 18th ed., 1992).
Animals. Adult female ND4 Swiss Webster mice (weight, 20 to 24 g) were used
for this study. The animals were anesthetized with 0.4 ml of 5% sodium pento-
barbital by intraperitoneal (i.p.) injection. The mice were housed in the Texas
Tech University Health Sciences Center Vivarium. The animals were treated in
accordance with protocol no. 96020-06, approved by the Animal Care and Use
Committee at Texas Tech University Health Sciences Center in Lubbock.
Thermal injury model. The mouse model of thermal injury of Stieritz and
Holder (28), as modified by Hamood (11), was used in this study. Briefly, the hair
was clipped from the backs of anesthetized mice, and the area was denuded with
a commercially available hair remover. The mice were securely placed into a
template with an opening of 4.5 cm by 1.8 cm to expose their shaved backs. A
nonlethal full-thickness thermal injury to the skin was induced by placing the
exposed back area to 90°C water for 10 s. Fluid replacement therapy consisting
of a subcutaneous (s.c.) injection of 0.8 ml of a 9% NaCl solution was adminis-
tered immediately following the burn. The mice were challenged by s.c. injection
of 100 ?l of the PAO1Rifinoculum (2 ? 102to 3 ? 102CFU) directly under the
anterior end of the burn. The phage cocktail was administered immediately after
the P. aeruginosa challenge.
During recovery from anesthesia, the mice were kept under warming lights and
observation. The cumulative mortality among the treatment groups was recorded
at 48 and 72 h postinfection (hpi). Lethargic animals were monitored every hour.
The surviving animals were killed at 96 hpi. Liver and spleen tissue samples were
removed from the animals, weighed, suspended in 2 ml of filter-sterilized PBS
(pH 7.2), and homogenized with sterile mortars and motor-driven Teflon pestles.
The numbers of PAO1Rifcells (CFU/g tissue) were determined by serial dilution
and plating on LB agar containing 80 mg/ml of rifampin. The titers (PFU/g
tissue) of the phages recovered from the tissues of mice were determined by the
soft agar overlay technique described above, following the addition of a few
drops of chloroform (per ml).
Pharmacokinetic studies. A phage cocktail inoculum (?3 ? 108/100 ?l per
inoculum) was administered to unwounded, noninfected mice i.p., intramuscu-
larly (i.m.). or s.c. Three animals from each treatment group were killed at 0.5,
12, 24, 36, and 48 h following phage injection. Briefly, blood, obtained by cardiac
puncture from anesthetized animals, was collected in tubes containing EDTA.
After blood collection, the anesthetized mice were euthanized by injection of
Fatal Plus (sodium pentobarbital), followed by cervical dislocation. Liver and
spleen tissue samples were removed from the animals. The tissues were weighed,
suspended in 2 ml of filter-sterilized PBS, and homogenized with sterile mortars
and motor-driven Teflon pestles. The phages in the tissues were enumerated as
described above and are expressed as PFU/gram of tissue.
Statistical analysis. Fisher’s exact test (Statview for Windows; SAS Institute,
Cary, NC) was used to determine the significance of the differences in survival
among the treatment groups and the differences in the distributions of the
phages to the tissues.
Protection studies. The ability of the P. aeruginosa-specific
phages to prevent P. aeruginosa infections was examined by use
of a modified mouse model of thermal injury, as described
above. A phage cocktail containing 1 ? 108PFU of each of
three different phages (3.0 ? 108PFU total) was administered
i.p., i.m., or s.c. to infected and uninfected wounded animals.
As a control for the virulence of the PAO1Rifinoculum, an-
other group of mice was injected with the bacterial inoculum
only (no phage). To examine the toxicity of the phages in
compromised animals, the wounded but noninfected groups
were injected with the phage cocktail (but not P. aeruginosa).
Animal deaths were recorded at 48 and 72 hpi.
The results of these experiments are presented in Table 1.
All of the thermally injured mice that were not infected with
PAO1Rifbut administered the phage cocktail survived, indi-
cating that the phage cocktail was not toxic to traumatized
mice. In the absence of phage there was a 94% rate of mor-
tality in the wounded infected mice in the first 72 hpi. When
the phages were administered i.m. or s.c., the rates of mortality
were reduced to 72% and 78%, respectively; and by sharp
contrast the rate of mortality was reduced to 12% when the
phages were delivered i.p. These results demonstrate that the
parenterally administered phages significantly increased sur-
vival in infected and wounded mice and that the relative pro-
TABLE 1. Protection studies: efficacy of phage therapy on P. aeruginosa infection of burn wounds
No. of survivors/total no. of mice (% survival)a
Expt 1Expt 2Expt 3 Combined exptb
48 hpi72 hpi 48 hpi 72 hpi48 hpi 72 hpi48 hpi 72 hpi
Phage by i.p. route only
Phage by i.m. route only
Phage by s.c. route only
Phage by i.p. route ? PAO1 infection
Phage by i.m. route ? PAO1 infection
Phage by s.c. route ? PAO1 infection
aThe cumulative survival was determined at 48 h and 72 hpi.
bThe average percent survival of animals in three experiments determined at 48 and 72 hpi.
cThe rate of survival for animals receiving the phage cocktail i.p. was significantly greater (P ? 0.0001) than that for untreated infected control animals at the same
dThe rate of survival for animals receiving the phage cocktail i.p. was significantly greater (P ? 0.01) than that for untreated infected control animals at the same
eThe rate of survival for animals receiving the phage cocktail i.p. was significantly greater (P ? 0.0026) than that for untreated infected control animals at the same
VOL. 51, 2007PHAGE THERAPY OF BURN WOUNDS 1935
tection afforded by the different routes of phage administration
was i.p. ? i.m. or s.c.
Of the wounded and infected animals receiving the phages
i.p., only 2 of the 17 animals had died by 72 hpi (at 53 hpi and
64 hpi, respectively). The surviving animals were killed at 96
hpi, and the numbers of PAO1Riforganisms detected from the
tissues of surviving animals was compared to the numbers from
the tissues of animals that died from the P. aeruginosa infec-
tion. The mean bacterial counts in the tissues of animals that
died were 1.53 ? 109CFU/g liver and 6.68 ? 107CFU/g
spleen. The mean bacterial counts in the tissues following
successful i.p. phage therapy were 5.26 ? 102CFU/gram liver
and 2.93 ? 102CFU/gram spleen. These results suggest that
the cause of death was the result of systemic P. aeruginosa
infection and that, as one might expect, successful phage ther-
apy correlates with a reduction in the PAO1Rifburden.
To determine if phage-resistant derivatives of PAO1Rif
emerged from the infected mice, we analyzed the PAO1Rif
isolates from the tissues of those mice that had died by 48 hpi
for their phage sensitivities. All isolates tested (?100) were
sensitive to each of the phages in the cocktail (data not shown).
Hence, the death of these animals was not the result of the
emergence of a phage-resistant derivative of the PAO1Rif
strain. The numbers of phages in the liver and spleen of ani-
mals that succumbed to P. aeruginosa infection in the first 48
hpi were also enumerated to determine if the phages had
multiplied. Considering the average weight of these organs (4.6
g and 2.27 g of liver and spleen, respectively) and assuming
100% phage recovery, each mouse harbored a minimum of
7.5 ? 109to 10 ? 109phages. This represents an increase of
approximately 20-fold over that administered to these mice
(?3 ? 108/mouse), indicating that the phages multiplied in
vivo, although obviously not to levels that were enough to save
Pharmacokinetic studies. In an attempt to determine why
delivery of the phages by the i.p. route was more efficacious
than delivery of the phages by the i.m. or the s.c. route for the
treatment of infected animals, we examined the pharmacoki-
netics of the phages introduced by the i.m., s.c., or i.p. route in
uninjured, uninfected animals. Three animals each from
groups receiving the phage cocktails i.p., i.m., or s.c. were killed
at 0.5, 12, 24, 36, and 48 hpi. The numbers of phages detected
per gram of liver and spleen and per milliliter of blood are
shown in Fig. 1. In each tissue examined, a consistent pattern
of the relative PFU levels after the administration of the
phages by the different routes was observed: i.p. ? i.m. ? s.c.
In the mouse model of thermal injury, 2 ? 102to 3 ? 102
PAO1Rifinjected at the burn site resulted in 83 to 100%
mortality by 48 hpi. Rumbaugh et al. have shown that the P.
aeruginosa organisms in such an infection proliferate and
spread systemically from skin to underlying tissues and that
within 24 hpi as many as 104PAO1RifCFU per gram of tissue
was detected in the liver and spleen (21). In this study we have
demonstrated that a single dose of a phage cocktail can effec-
tively decrease the rate of mortality due to P. aeruginosa in-
fection of burn wounds in the mouse model of thermal injury.
This protection was shown to be the result of a significant
decrease in the numbers of P. aeruginosa organisms found in
the successfully treated animals, indicating that the bacterial
viruses used were able to locate and kill PAO1Rifbefore the
animal succumbed to bacteremia and septic shock. However, it
was also found that not all infected animals which were treated
with the phages survived and that the route of phage admin-
istration was particularly important to the efficacy of the
treatment, with the i.p. route providing the most significant
protection (87%) of the routes tested (Table 1).
It was also found that the P. aeruginosa phages had multi-
plied in mice that had died of infection and that phage-resis-
tant P. aeruginosa strains were not recovered from these ani-
mals. These results suggest that the use of a phage cocktail
containing phages that use different receptors may have pre-
vented the emergence of phage-resistant mutants and that the
therapeutic phages had found their host (PAO1Rif) and mul-
tiplied, but apparently not in sufficient time and/or in sufficient
numbers to prevent mortality. Hence, the differences in the
efficacies of the different routes of phage administration may
be due to the rate and dose of phage delivery to their targets.
This explanation is somewhat supported by the observation
that the P. aeruginosa phages administered by the i.p. route
were delivered at a higher dose, were delivered earlier, and
were delivered for a more sustained period of time to the
examined tissues of a mouse (Fig. 1) than the phages delivered
by the s.c. or i.m. route.
It should finally be pointed out that the mouse model of
thermal injury is a very stringent test of phage therapy for a
systemic infection. Highly virulent PAO1Rifcells injected s.c.
beneath the burn wound, allowing the organisms to proliferate
very quickly and spread systemically to cause septic shock and
death in a relatively short period of time (24 to 48 h). By
comparison, the rates of proliferation and systemic dissemina-
tion from natural infections acquired at burn wound surfaces
are usually much slower. Furthermore, in most human burn
wounds, infection occurs after admission to a hospital or burn
ward and is often caused by a hospital-associated strain. The-
oretically, such infections could be more conducive to phage
therapy/prophylactics than would P. aeruginosa infections in
the animal model tested here because of the chronic nature of
the infection. On the other hand, some chronic wounds are
populated by biofilms, which may be more difficult to treat with
phage and/or may require different types of therapeutic phage
than that used for the treatment of systemic infections.
Chronic infection may require prolonged treatment with
phage, which may in turn select for phage resistance and in-
FIG. 1. Pharmacokinetics of the phages in noninfected mice. A cocktail containing ?3 ? 108phage was injected i.p., i.m., or s.c. into uninjured,
uninfected mice. Groups of mice (n ? 3) were killed at 0.5 h hpi, 12 hpi, 24 hpi, 36 hpi, or 48 hpi. Tissues were removed and the numbers of PFU
were determined by serial dilution and plating on PAO1Rif. (A) PFU/gram liver; (B) PFU/gram spleen; (C) PFU/ml blood. Values are means ?
standard errors of the means.
VOL. 51, 2007PHAGE THERAPY OF BURN WOUNDS1937
duce an immune response, which could reduce the therapeutic
value of phage treatment, although phages, unlike antibiotics,
evolve with their host(s). Obviously, more detailed studies ex-
amining the effect of the phage dosage, the routes and timing of
phage administration, the pharmacokinetics and tissue tropism of
the phages used, as well as determination of the phenotypic traits
of the most effective therapeutic phages for particular types of
infections will be needed to determine if phage therapy will pro-
vide a much needed alternative/supplement for the treatment
of bacterial infections. However, with that said, recent, well-
controlled animal studies, which have successfully applied phage
therapy to multiple types of bacterial infections, have spawned
new enthusiasm for an old idea (3–7, 10, 14, 19, 23–26, 33–35),
and the FDA has recently approved phase I trials for the use of
phage to treat bacterial infections of diabetic foot ulcers (R.
Wolcott personal communication and http://sanjose.bizjournals
We thank Abdul Hamood and Kendra Rumbaugh for their moral
and technical support, John Griswold for providing space and equip-
ment to accomplish thermal injuries, and Ben Burrows and Probhjit
Chadha-Mohanty for critical reading of the manuscript.
This work was supported in part by a Texas Tech University School
of Medicine seed grant (to J.A.F.) and grants from the Texas Tech
University Biology Department (to M.V.) and the Texas Tech Univer-
sity Graduate School (to M.V.).
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1938MCVAY ET AL.ANTIMICROB. AGENTS CHEMOTHER.