Full-thickness porcine burns infected with Staphylococcus

aureus or Pseudomonas aeruginosa can be effectively

treated with topical antibiotics
David M. Tsai, MD1; Lauren E. Tracy, MD1; Cameron C. Y. Lee, BS1; Florian Hackl, MD1;
Elizabeth Kiwanuka, MD, PhD1; Raquel A. Minasian, BS1; Andrew Onderdonk, PhD2;
Johan P. E. Junker, PhD1; Elof Eriksson, MD, PhD1; E. J. Caterson, MD, PhD1
1. Division of Plastic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts,
2. Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

Reprint requests: ABSTRACT

E.J. Caterson MD, PhD, Brigham and
Women’s Hospital, Division of Plastic
Surgery, 75 Francis Street, Boston,
MA 02115.
Tel: 617-732-7409;
Fax: 617-732-6387;
Email: ecaterson@partners.org
Funding: No external funding to disclose
Manuscript received: January 20, 2015
Accepted in final form: January 17, 2016
DOI:10.1111/wrr.12409
Burn and blast injuries are frequently complicated by invasive infections, which
lead to poor wound healing, delay in treatment, disability, or death. Traditional
approach centers on early debridement, fluid resuscitation, and adjunct
intravenous antibiotics. These modalities often prove inadequate in burns, where
compromised local vasculature limits the tissue penetration of systemic
antibiotics. Here, we demonstrate the treatment of infected burns with topical
delivery of ultrahigh concentrations of antibiotics. Standardized burns were
inoculated with Staphylococcus aureus or Pseudomonas aeruginosa. After
debridement, burns were treated with either gentamicin (2 mg/mL) or
minocycline (1 mg/mL) at concentrations greater than 1,000 times the minimum
inhibitory concentration. Amount of bacteria was quantified in tissue biopsies
and wound fluid following treatment. After six days of gentamicin or
minocycline treatment, S. aureus counts decreased from 4.2 to 0.31 and 0.72 log
CFU/g in tissue, respectively. Similarly, P. aeruginosa counts decreased from
2.5 to 0.0 and 1.5 log CFU/g in tissue, respectively. Counts of both S. aureus
and P. aeruginosa remained at a baseline of 0.0 log CFU/mL in wound fluid for
both treatment groups. The findings here demonstrate that super-therapeutic
concentrations of antibiotics delivered topically can rapidly reduce bacterial
counts in infected full-thickness porcine burns. This treatment approach may aid
wound bed preparation and accelerate time to grafting.

Burn and blast injuries are devastating consequences of
modern military conflicts.1–5 Successful management
hinges on timely, aggressive wound care that focuses on
decreasing contamination, removing necrotic tissue, and
initiating antimicrobial prophylaxis. Invariably, these
wounds are subject to infections, which cause profound
morbidity and mortality.6–10 Thus, the primary goal in
treating these injuries should be infection prevention.
Unfortunately, current treatment modalities frequently
prove inadequate in this regard. Moreover, these injuries
often occur in the field, where no frontline measures exist
that can reliably curtail invasive infections and prevent
subsequent sepsis.11,12 With the growing threat of terrorist
attacks on civilian soil and the increased incidence of
civilian casualties in modern conflicts, the need for a safe
and effective way to prevent and treat burn infections has
become strikingly important to the population as a whole.
Unlike systemic antibiotic prophylaxis, topical antimi-
crobials have become well established as an adjunct to
early excision and grafting.13–15 They are exceptionally
advantageous in the context of burns, which are encum-
bered by devitalized tissue and diminished blood supply.
By administering antimicrobials directly to a wound,
356 Wound Rep Reg (2016) 24 356–365 V C 2016 by the Wound Healing Society
higher concentrations can be reached without the need for
a healthy, intact vascular network. The risk of adverse
systemic side effects is also dramatically diminished,
making topical antimicrobials much more appealing than
their systemic counterparts. Without their use in the
immediate postburn period, burn wounds are quickly
colonized by staphylococcus, streptococcus, and gram
negative species. Of these, Staphyloccocus aureus, Pseu-
domonas aeruginosa, and Klebsiella pneumoniae have the
most potential to cause invasive wound infections, and S.
aureus and P. aeruginosa are most frequently associated
with bacteremia.16–20
Silver sulfadiazine, mafenide acetate, silver nitrate are
some of the most commonly used topical antimicrobials
today for burn care.14,15,21–23 These agents exist as creams
or aqueous solutions that are applied with dry or moist
dressings, which often require multiple dressing changes.24
Despite the proven efficacy in decreasing burn infections,
using these agents in the correct manner can strain a
facility’s time and resources.11,12 Further, these topical
antimicrobials need to be applied immediately and are
effective only after proper cleansing and debridement.12
This level of wound care is unrealistic in the battlefield,

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Tsai et al. Topical antibiotics for treatment of infected burns

This study is in response to the need for a treatment

Figure 1. Polyurethane wound enclosure device. Shown
here is a schematic of the polyurethane wound enclosure
device. A circumferential adhesive rim permits complete
enclosure of a wound, and thereby establishes a protected,
incubator-like environment. A self-sealing injection port
allows easy sampling of wound fluid or delivery of liquid sol-
utions such as antibiotics, growth factors, or analgesia in
precise amounts and concentrations.
and first responders are often not equipped with these
agents. Finally, these topical antimicrobials have no proven
ability to treat burn wounds plagued with an existing
infection.21,25 In these cases, a shift to systemic antibiotic
therapy is often warranted.26 Nonetheless, systemic agents
prove less than ideal because they generally exhibit poor
tissue penetration, especially in burns where local wound
vasculature is compromised. Higher doses are thus needed
to reach therapeutic concentrations in tissue, which
unavoidably increases the risk of adverse side effects asso-
ciated with systemic antibiotics such as nephropathy and
disruption of GI flora.
modality that can safely and effectively decrease contami-
nation of infected burn wounds. Building upon our earlier
work on a topical antibiotic delivery system that uses a
polyurethane wound enclosure device or “wound chamber”
(Figure 1),27–31 we extended the treatment methodology to
full-thickness porcine burns infected with S. aureus or
P. aeruginosa. We hypothesized that this system would be
effective in rapidly reducing the bacterial burden of
infected burn wounds. Moreover, by correlating infection
clearance over time with changes in levels of inflammatory
cytokines, we sought to compare antibiotics and pinpoint
an optimal duration of treatment with respect to balancing
rapid wound decontamination with a possible delay of
local wound healing.
MATERIAL AND METHODS
Animals
All animal procedures performed in this study received
prior approval from the Harvard Medical Area Standing
Committee on Animals (Protocol # 00693), and conformed
to federal statutes and regulations regarding animal use.
Nine female Yorkshire pigs (Parson’s Farm, Hadley, MA)
weighing between 50 and 65 kg were used in the study.
The animals were socially housed and acclimatized for 72
hours prior to any procedures.
Burn device
We used a custom burn device (Figure 2) to create stand-
ardized full thickness burn wounds (Figure 3). It consists
of an aluminum disk that is insulated with cork on all
sides except the base, which comes into direct contact
with the skin. A probe is embedded in the device to

Figure 2. Custom aluminum burn device. (Left) An aluminum disk, 2 cm in diameter, is insulated with cork on all sides except
the base, which comes into direct contact with the skin. An embedded probe allows accurate measure of the temperature
drop upon application of device to skin. This allows precise calculation of the amount of heat energy transferred for each
wound, ensuring standardized full-thickness burns. (Right) The device is heated up to 230 8C and applied to the skin until the
temperature dropped to 180 8C. To ensure equal force of application among all wounds during the burn creation, a custom
plunger device calibrated to 10 Newton of force is used to apply the burn device to the skin. This helps eliminate operator var-
iance and allows comparison of wounds from the same pig, from different pigs, and from other investigators.

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Topical antibiotics for treatment of infected burns
Figure 3. Standardized full-thickness burns. Histological
image of the depth of burn as demonstrated by the homoge-
nization of collagen fibrils. The burns extended through the
epidermis, dermis, and parts of the subcutaneous adipose
tissue. Immediately after burn, each wound was inoculated
with 108 CFU/mL of bacteria at a depth of 8 mm. Excisional
debridement was performed at approximately the level of
the panniculus carnosus.
accurately record the drop in temperature upon application
of the device to the skin. This allows precise calculation
of the amount of heat energy transferred for a defined sur-
face area during the burn (32 calories/cm2). The device is
heated up to 230 8C and applied to the skin until the tem-
perature dropped to 180 8C. To ensure equal force of appli-
cation among all wounds during the burn creation, a
custom plunger device calibrated to 10 Newton of force is
used to apply the burn device to the skin (Figure 2). This
helps eliminate operator variance and allows comparison
of wounds from the same pig, from different pigs, and
from other investigators.
Porcine burn model
Anesthesia was induced with intramuscular administration
of 4.4 mg/kg zolazepam and tiletamine (Telazol; Fort
Dodge Veterinaria, Madrid, Spain) and 2.5 mg/kg xylazine
358
Tsai et al.
(Xyla-Ject; Phoenix Pharmaceutical, St. Joseph, MO). Gen-
eral anesthesia was maintained with 2% isoflurane (Hos-
pira, Lake Forest, IL) and oxygen via endotracheal
intubation. A pulse oximeter was used to monitor oxygen
saturation and heart rate. Temperature was maintained at
38 8C with heating blankets.
The dorsum of the pig was waxed and shaved. Skin was
disinfected with 10% povidone-iodine scrub (Purdue
Pharma, Stamford, CT) and 70% isopropanol (Aaron
Industries, Clinton, SC). Up to 26 full thickness circular
burns, 2 cm in diameter were created on each pig using
the burn device described above. The depths of burns were
verified histologically (Figure 3). With a 32-gauge needle,
each wound was inoculated with 108 colony-forming units
(CFU) of the chosen pathogen diluted in 1 mL saline, at a
depth of 8 mm (Figures 3 and 4A), in the subeschar plane.
To prevent cross-contamination and confounding, each pig
was only inoculated with one type of microbe, either S.
aureus (ATCC 12600) or P. aeruginosa (ATCC 27853).
The burns were subsequently covered with sterile gauze
and leukotape (BSN Medical, Hamburg, Germany), and
allowed to incubate for 3 days (Figure 4B).
On day 3 postburn, the eschar and necrotic tissue were
debrided from all wounds in a standardized fashion, at the
level of the panniculus carnosus and beyond the zone of
coagulation at the zone of stasis with a standardized sized
cutting tool (Figure 4C). Wounds were divided into those
receiving dry treatment and topical treatment. For those
receiving the latter, polyurethane enclosure devices were
applied (Figure 4D), and randomly assigned to one of two
groups using a random number generator (http://www.
random.org): one receiving 10 mL of saline, and the other
receiving 10 mL of the chosen antibiotic (2 mg/mL gentami-
cin or 1 mg/mL minocycline, >1,000 times minimum inhibi-
tory concentration). Concentrations were chosen to roughly
equate MICs and to allow comparison with previous studies.
Each pig received only one antibiotic (gentamicin or minocy-
cline) during the length of the experiment. For accuracy and
consistency, devices were emptied daily, sampled, and rein-
jected with 10 mL of the chosen treatment solution; effec-
tively diluting “bona fide” wound fluid by a factor of 10.
This was later corrected in subsequent analyses.
Daily fluid was collected from the polyurethane devices
with an 18-gauge needle and flash frozen for later analysis.
On days 3, 6, and 9 after burn creation, tissue was sampled
at an appropriate depth using a 6 mm punch biopsy, making
sure to obtain a small portion of healthy underlying tissue.
Specimens were placed in dry, sterile cryogenic vials and
flash frozen for later analysis. Biopsied wounds were subse-
quently excluded from the remainder of the experiment.
Wounds were treated as separate entities and as N, with at
least 6 wounds for each time point and treatment group.
Quantitative bacterial cultures
Tissue punch biopsies and fluid samples were collected as
described above. Analysis was performed by the Brigham
and Women’s Hospital Clinical Microbiology Laboratory.
Quantitative bacteriology was performed to assess infec-
tions levels of S. aureus and P. aeruginosa remaining in
tissue and fluid after excisional debridement, prior to sub-
sequent treatment. Counts were then subsequently per-
formed to quantify infection clearance on days 3, 6, 9.
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Tsai et al. Topical antibiotics for treatment of infected burns

Figure 4. Inoculation, debridement, and treatment. (A) On day 0, after the burns were created, Jackson’s zones of coagula-
tion, stasis, and hyperemia were immediately evident. A 27-gauge needle with a custom needle-stopper was then used to
inoculate the burns with 108 CFU/mL of bacteria at a depth of 8 mm. (B) Appearance of burns after 3 days of incubation.
(C) Wounds after excisional debridement. (D) Application of polyurethane device on debrided wound. Devices were then filled
with 10 mL of either saline or antibiotics.

Samples were serially diluted, plated on blood agar plates, RESULTS

and examined for S. aureus and P. aeruginosa growth.
Peripheral blood analysis
At appropriate time points, venous blood was drawn from
the ear veins and sent to the Clinical Laboratory at Brig-
ham and Women’s Hospital for analysis. Blood counts for
white blood cells, red blood cells, hemoglobin, and hemat-
ocrit were determined. For gentamicin-treated pigs, serum
concentrations of gentamicin were assessed to monitor sys-
temic uptake and risk of toxicity.
Inflammatory mediators
Fluid samples from the polyurethane devices were diluted, acti-
vated, and analyzed with a sandwich enzyme linked immunosor-
bent assay using kits specific for porcine IL-1b (DNF0611051),
IL-6 (CBA0413011), TNF-a (EKQ0312011; R&D Systems,
Minneapolis, MN) per manufacturer’s protocol. Reactions were
stopped with 2M H2SO4 (Sigma Aldrich, St. Louis, MO) and
plates read with a SpectraMax M5 Microplate Reader and Soft-
Max Pro software (Molecular Devices, Sunnyvale, CA).
Statistical analysis
Statistical analysis was performed using GraphPad Prism
6.0 (GraphPad, La Jolla, CA). Data is presented as mean 6
SEM. Statistical comparisons were performed using a two-
way ANOVA with a Bonferroni posttest. p-Values under
0.05 were considered significant.
Reduction of S. aureus levels in tissue
After excisional debridement on day three, an infection
level of 4.2 6 0.3 log CFU/g remained in the wound tissue.
Treatment with gentamicin at 2 mg/mL (>1,000 times
MIC) further decreased S. aureus levels to 0.84 6 0.8 log
CFU/g (n 5 6) by day 6 (after 3 days of treatment) and
0.31 6 0.3 log CFU/g (n 5 9) by day 9 (after 6 days of
treatment; Figure 5). Treatment with minocycline at 1 mg/
mL (>1,000 times MIC) decreased S. aureus counts to
3.2 6 0.8 log CFU/g (n 5 8) and 0.72 6 0.7 log CFU/g
(n 5 6) by day 6 and 9 (after 3 and 6 days of treatment),
respectively. Levels in saline-treated tissue increased to
7.8 6 0.2 log CFU/g (n 5 12) by day 6 and subsequently
decreased to 6.9 6 0.2 (n 5 16) by the terminal endpoint,
day 9. In dry-treated wounds, levels of S. aureus increased
to 5.7 6 0.5 log CFU/g (n 5 11) by day 6 and decreased to
5.0 6 0.4 log CFU/g (n 5 14) by the terminal endpoint.
Compared with saline treatment and dry treatment, the dif-
ferences observed with the antibiotic treated groups were
statistically significant (p < 0.0001).
Reduction of S. aureus levels in wound fluid
S. aureus levels in wound fluid from both gentamicin-
treated wounds and minocycline-treated wounds remained
at the baseline of 0.0 6 0.0 log CFU/mL from the start of
treatment on day 3 to the terminal endpoint, day 9

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Topical antibiotics for treatment of infected burns Tsai et al.

Figure 5. Quantitative bacterial cultures in wound tissue. Tissue biopsies from wounds infected with S. aureus (left) and
P. aeruginosa (right) and treated with either gentamicin or minocycline were analyzed for amount of bacteria, in CFU, per
gram of tissue. Treatment with saline or dry dressings served as controls. Both topical administration of gentamicin and mino-
cycline significantly reduced bacterial counts at all time points; whereas bacterial counts were increased in the control groups.
Error bars represent SEM.

Reduction of P. aeruginosa levels in tissue

(Figure 6). In contrast, wound fluid from saline-treated
wounds increased from the baseline of 0.0 6 0.0 log
CFU/mL to 7.14 6 0.3 log CFU/mL, and 8.25 6 0.2 log
CFU/mL by day 6 and 9, respectively (Figure 6). The dif-
ference was statistically significant (p <0.0001).
An infection level of 2.5 6 0.4 log CFU/g remained in tis-
sue after excisional debridement three days after burn crea-
tion. Treatment with gentamicin at 2 mg/mL (>1,000
times MIC) reduced levels to 1.6 6 1.6 log CFU/g (n 5 7)

Figure 6. Quantitative bacterial cultures in wound fluid. Fluid samples from wounds infected with S. aureus (left) and
P. aeruginosa (right) and treated with either gentamicin or minocycline were analyzed for amount of bacteria, CFU, per milli-
meter of fluid. Treatment with saline served as controls. No fluid was collected with dry dressings. Addition of either gentami-
cin or minocycline rapidly reduced bacterial counts in fluid compared with saline treatment. Error bars represent SEM.

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Tsai et al.
by day 6 (after 3 days of treatment) and to 0.0 6 0.0 log
CFU/g (n 5 7) by day 9 (after 6 days of treatment). Treat-
ment with minocycline at 1 mg/mL (>1,000 times MIC)
decreased initial levels to 1.5 6 1.9 log CFU/g (n 5 7) and
1.5 6 2.1 log CFU/g (n 55) by day 6 and 9 (after 3 and 6
days of treatment), respectively (Figure 5). In contrast,
saline-treated wounds increased to 5.9 6 1.4 log CFU/g
(n 5 14) and 7.2 6 1.3 log CFU/g (n 5 14) by day 6 and 9,
respectively. In dry-treated wounds, levels increased to
5.2 6 1.1 log CFU/g (n 5 12) by day 6 and increased
slightly to 5.4 6 2.0 log CFU/g (n 5 12) by the terminal
endpoint, day 9. Again, compared with saline treatment
and dry treatment, the differences observed with gentami-
cin treatment and minocycline treatment were statistically
significant (p <0.0001).
Reduction of P. aeruginosa levels in wound fluid
P. aeruginosa levels in wound fluid from gentamicin-
treated wounds and minocycline-treated wounds remained
at the baseline of 0.0 6 0.0 log CFU/mL from the start of
treatment on day 3 to the terminal endpoint, day 9 (Figure
6). In contrast, wound fluid from saline-treated wounds
increased from the baseline of 0.0 6 0.0 log CFU/mL to
4.9 6 0.2 log CFU/mL and 6.1 6 0.6 log CFU/mL by day
6 and 9, respectively. The difference was statistically sig-
nificant (p < 0.0001).
Systemic uptake
Serum concentrations of gentamicin were undetectable
using the in-house assay (<0.4 mg/mL). No signs of sys-
temic toxicity were observed in any pigs.
Inflammatory response
On day 6, in S. aureus infected burns, levels of IL-6 and
TNF-a were significantly lower in minocycline-treated and
gentamicin-treated wounds, respectively, compared with
saline controls (Figure 7). On day 9, levels of IL-1b were
significantly higher in gentamicin-treated wounds com-
pared with saline controls. There was no significant differ-
ence observed between gentamicin and minocycline
groups.
In P. aeruginosa infected wounds, levels of Il-6 and
TNF-a were significantly lower in both gentamicin-treated
and minocycline-treated wounds compared with saline con-
trols on day 6 (Figure 7). Levels of IL-1b in minocycline-
treated wounds were significantly increased by day 9 com-
pared with both saline controls and gentamicin-treated
wounds. Conversely, IL-6 levels were significantly
decreased in minocycline-treated wounds compared to both
saline controls and gentamicin-treated wounds on day 9.
Levels of Il-6 in gentamicin-treated wounds were signifi-
cantly increased compared with saline controls on day 9.
Wound bed preparation
After six days of treatment, the antibiotic-treated wounds
exhibited red, healthy granulation tissue, which appeared
ready for skin grafting (Figure 8A,B). In comparison, the
saline-treated wounds were covered with what appeared to
be a layer of biofilm, and exhibited erythema and indu-
ration. Unsurprisingly, these wounds demonstrated high
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Topical antibiotics for treatment of infected burns
levels of infection when biopsied (Figure 8C). The dry-
treated wounds had a slightly better appearance than
saline-treated wounds; however, bacteria levels in these
wounds still exceeded 105 CFU/g at the terminal endpoint
(Figure 8D).
DISCUSSION
This study demonstrates the effective treatment of infected
full-thickness porcine burns with a topical antibiotic treat-
ment platform. Building upon the principles and advan-
tages of topical antimicrobials, this modality improves on
existing formulations. In particular, the polyurethane
enclosure device can establish a controlled incubator-like
wet environment, which is conducive to optimal wound
healing and allows direct delivery of antibiotic solutions
(Figure 1). By diluting a typical single IV antibiotic dose
in a small volume in contrast to systemic distribution, we
can achieve concentrations in orders of magnitude greater
than intravenous delivery (>1,000 times MIC). This allows
for rapid reduction of bacterial burden while isolating and
protecting the wound from both the environment and
potential contamination from other wounds. From a practi-
cal standpoint, the device has a self-adhesive rim that
facilitates application to the wound. The injection port
allows timely administration of antibiotics that can be
reconstituted with water, or analgesic solutions to provide
immediate pain relief.
In recent years, several other topical delivery strategies
have been described. Collectively, these efforts represent a
paradigm shift in the way clinicians are thinking about
treating and managing wounds. For instance, irrigation-
based systems exist as described by Svedman,32 Kinetic
Concepts Incorporated,33 and Zamirowski,34 in which anti-
microbials are delivered via pulsatile irrigation. Although
promising, these systems have difficulty delivering precise
amounts of antibiotics since it is difficult to gauge how
much is actually absorbed. Other strategies have included
antimicrobial polymers in the form of dressings, sponges,
or patches. These polymers are engineered with hydrocol-
loids, alginates, collagen, and others.35,36 Although some
studies have been encouraging, a shared limitation among
these different strategies is that they are inherently fixed
systems, and lack the capability of altering the antimicro-
bial agent or titrating the dose. In comparison, our delivery
system enables the clinician to precisely administer an
antibiotic in varying concentrations at one’s discretion.
Further, the injection port allows fluid sampling and
removal or exchange of fluid. This is conducive to initial
broad-spectrum antibiotic administration and a facile
switch to culture-directed narrow-spectrum antibiotics.
To evaluate our treatment system, we developed a por-
cine model to study infected full-thickness burns. Using an
aluminum burn device, we delivered equivalent amounts
of heat energy to each wound under equal pressure to
ensure full-thickness burns were standardized across all
animals (Figure 2). Subsequently, we tested the treatment
on burns infected with either S. aureus or P. aeruginosa,
two of the most common pathogens associated with bacter-
emia and invasive burn infections.16–20 To properly assess
bacterial clearance, we performed tissue biopsies, which
has been the historical standard in diagnosing invasive
burn infections and correlating counts with burn
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Topical antibiotics for treatment of infected burns Tsai et al.

Figure 7. Levels of inflammatory cytokines in wound fluid. Fluid samples from wounds infected with S. aureus (left) and
P. aeruginosa (right) and treated with either gentamicin or minocycline were analyzed for concentrations of IL-1b, IL-6, and
TNF-a. Levels of inflammatory markers were generally decreased after 3 days of treatment relative to saline control. This was
primarily noted in IL-6 and TNF-a levels. After 6 days of treatment, some inflammatory markers increased in antibiotic-treated
groups relative to saline control such as IL-1b and IL-6. Error bars represent SEM.

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Figure 8. Wound bed preparation six
days posttreatment. Clinical appear-
ance of burns after treatment with
gentamicin (A), minocycline (B),
saline (C), and dry dressings (D).
sepsis.25,37,38 We also assessed bacterial levels in wound
fluid, since high levels of bacteria in fluid can often serve
as a continuing inoculant in burn wounds, and in a health-
care environment can act a route of nosocomial transmis-
sion or cross-contamination among wounds.25 Additionally,
since gentamicin is commonly associated with systemic tox-
icity, serum concentrations were monitored and served as a
measure for systemic uptake for our delivery system.
Our results establish the capability of ultrahigh concen-
trations of topical antibiotics to dramatically reduce bacte-
rial counts in infected burn wounds. Despite the standard
practice of excisional debridement, there remained consid-
erable levels of bacteria in the tissue, up to 104 CFU/g in
S. aureus infected burns, serving to illustrate that debride-
ment alone is insufficient and adjunct antibiotics are neces-
sary. Subsequently, bacterial counts in tissue from S.
aureus infected burns dropped by 10,000 fold after 6
days of treatment with topical gentamicin or minocycline.
In contrast, bacterial counts dramatically increased in the
control groups, and by the terminal endpoint, there was
100,000-fold difference in bacterial counts between con-
trol groups and treatment groups. In P. aeruginosa infected
burns, less bacteria remained in tissue after initial debride-
ment, but by the terminal endpoint in dry and saline
control groups, bacteria levels increased to 105 and 107
CFU/g, respectively, compared with negligible levels in
gentamicin and minocycline groups. Overall, this data indi-
cates that this treatment method is not only effective for
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Topical antibiotics for treatment of infected burns
rapidly reducing high bacterial burden in burns but can
doubly serve as a prophylactic measure. The ability to
achieve two crucial aims of burn management simultane-
ously is a powerful advantage when compared with current
modalities such as topical antimicrobials and systemic anti-
biotics, which can only accomplish one.
One of the key benefits of reducing bacterial burden is
wound bed preparation, which is in line with the ultimate
goal of skin closure. It is well known that infections can
impede wound healing and graft take, and increase scarring.
As shown in Figure 8, antibiotic treated wounds exhibited a
healthy wound bed that appeared prime for skin grafting.
As an additional measure of the efficacy of topical anti-
biotics, we assessed relative changes in levels of inflam-
matory markers after 3 and 6 days of treatment. We
acknowledge these changes to be a complex interplay of
five factors: (1) initial burn, (2) trauma from excisional
debridement, (3) type of pathogen, (4) bacterial levels, and
(5) type of antibiotic administered. Since all wounds were
subjected to the same degree of burn and surgical trauma,
it is reasonable to conclude that the bacteria and antibiotics
are primarily responsible for any differences in cytokine
levels. Although difficult to interpret in isolation, when
viewed as a whole we recognize a trend in which the lev-
els of inflammatory markers were decreased after 3 days
of treatment relative to saline control (Figure 7). This can
be primarily noted in IL-6 and TNF-a levels. Ostensibly,
these differences can be attributed to the respective
363
Topical antibiotics for treatment of infected burns
reduction in bacterial counts. After 6 days of treatment,
however, some inflammatory markers increased in
antibiotic-treated groups relative to saline control such as
IL-1b and IL-6. The challenging question to answer is
whether this is a consequence of local inflammation and
cytotoxicity caused by prolonged exposure to ultrahigh
concentrations of antibiotics or if the increased levels are
due to a mass release of products from lysed bacteria that
subsequently triggers a surge in inflammatory cytokines.
Future work focused on further detailing the inflammatory
response will undoubtedly unveil more about these intri-
cate interactions. Within this study, however, we can view
these inflammatory markers in conjunction with levels of
bacteria on corresponding days. By day 6, most of the bac-
terial contaminants have been dramatically reduced. Tak-
ing this into account, we aim to titrate or cease antibiotics
in the future after 3 days of treatment as a way to balance
bacterial reduction with any inflammatory response that
may potentially impede healing. Nevertheless, we believe
the benefits of bacterial reduction outweigh the temporary
delay in wound healing, as it can profoundly prepare the
wound bed for grafting.
With the application of this treatment to large burns, we
expect the toxicity to remain minimal relative to systemic
antibiotics, since the uptake is limited by the compromised
vasculature of burns. Higher concentrations can then be
used, as we have exhibited in this study, and greater thera-
peutic levels reached at a much lower risk of toxicity.
Nevertheless, the use of any antibiotics regardless of for-
mulation should be accompanied with vigilant monitoring
and should be guided by serum levels. To further mitigate
risk, the concentrations of the topical antibiotics are easily
titrated.
In conclusion, ultrahigh concentrations of topical genta-
micin or topical minocycline can rapidly reduce the bacte-
rial burden of full-thickness porcine burns infected with
either S. aureus or P. aeruginosa. The polyurethane enclo-
sure device permits precise, controlled delivery while min-
imizing the risk of systemic toxicity. Treatment with this
proposed methodology may aid wound bed preparation
and accelerate time to subsequent grafting.
ACKNOWLEDGMENTS
We would like to thank Andrea Dubois for her exceptional
technical assistance and Anders Carlsson and Olle Eng-
strom for their contributions to the porcine experiments.
Conflict of Interest/Disclosure Statement: Dr. Eriksson
has filed a number of patents related to the wound chamber
platform technology, and is a member of a LLC that deals
with wound healing. The remaining authors have no com-
mercial associations or financial disclosures that might
pose or create a conflict of interest.
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