ORIGINAL ARTICLE
Intrawound Vancomycin Powder Reduces Bacterial Load in
Contaminated Open Fracture Model
Cyrus Caroom, MD,* Dustin Moore, MD,* Nithya Mudaliar, MS,† Craig Winkler, MD,*
Jefferson Murphree, MD,* Ian Ratheal, MD,* Michael Fry, BS,* Mark Jenkins, MD,*
Jessica Tullar, PhD, MPH,‡ and Abdul Hamood, PhD†
Objectives: To compare the effectiveness of both vancomycin
powder and antibiotic bead placement to irrigation and debridement
alone in prevention of infection in a contaminated open fracture
model in rats.
Downloaded from http://journals.lww.com/jorthotrauma by BhDMf5ePHKbH4TTImqenVEyMrwFXE1ZVhn6Xws/6sEV8j3ijuP0a41JtAjeHrFdT on 11/10/2018
Methods: In a previously described model of contaminated open
fractures, 45 rats had simulated open fractures created, stabilized,
and contaminated with Staphylococcus aureus. They were then trea-
ted 6 hours later with 3 interventions: irrigation and debridement
alone (control group) or in combination with placement of polymeth-
yl methacrylate beads containing vancomycin and tobramycin pow-
ders (antibiotic bead group) or placement of 10 mg of intrawound
vancomycin powder (powder group). Rats were allowed to recover
and then killed 14 days later for harvest of femurs and plates. Femurs
and plates were both incubated overnight, and bacterial colonies
were counted in each group for comparison.
Results: Quantitative counts of bacteria in bone showed signifi-
cantly reduced growth in both bead and powder groups when
compared with control group (P , 0.0001). Quantitative counts of
bacteria in plates showed significantly reduced growth in both bead
and powder groups when compared with control group (P , 0.0003;
0.029). No significant differences were seen in bacterial growth
between bead and powder groups for either bones (P = 0.13) or
plates (P = 0.065).
Conclusions: When compared with irrigation and debridement
alone, placement of intrawound vancomycin powder significantly
decreased bacterial load in a contaminated open fracture model in
rats similar to placing antibiotic beads. This may provide an
additional adjuvant treatment that does not require a secondary
surgery for bead removal.
Accepted for publication June 8, 2018.
From the Departments of *Orthopaedic Surgery; †Immunology and Molecular
Microbiology, Texas Tech University Health Sciences Center, Lubbock,
TX; and ‡Institute for Health Policy, University of Texas Health Science
Center School of Public Health, Houston, TX.
C. Caroom received a grant for this study from AO trauma (Kathryn Cramer
Career Development Award Project no: IACUC 15017). C. Caroom and
M. Jenkins are consultants for DePuy Synthes for unrelated work. The
remaining authors report no conflict of interest.
Presented in part at the Annual Meeting of the Orthopaedic Trauma
Association, October, 11, 2017, Vancouver, BC.
Reprints: Cyrus Caroom, MD, Texas Tech University Health Sciences
Center, 3601 4th St, TTUHSC Mail Stop 9436, Lubbock, TX 79430
(e-mail: cyrus.caroom@ttuhsc.edu).
Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.
DOI: 10.1097/BOT.0000000000001261
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Key Words: infection, vancomycin, open fracture
(J Orthop Trauma 2018;32:538–541)
INTRODUCTION
Open fractures are relatively common and the treatment
of these is often associated with surgical site infections
(SSIs).1–3 SSI rates have been reported from 2% to 50%.4
The treatment of these infections more than doubles the cost
compared with patients without infection.5 Prompt treatment
both with irrigation and debridement and prompt administra-
tion of intravenous antibiotics have shown to decrease infec-
tion rates. However, often due to both the severity of
contamination and the high energy nature of injuries, these
wounds have compromised vascularity. Therefore systemic
antibiotics are often not enough to eradicate an initial infec-
tion, leading to disastrous consequences for the patient.
Intrawound local antibiotic delivery has been shown to
decrease infection rates in open fractures, decreasing infection
rates from 8%–12% to 3%–5%.4 These are traditionally deliv-
ered in the form of polymethyl methacrylate (PMMA) beads
that require removal before wound closure or at a later sur-
gery.6–8
Local vancomycin powder applied to clean wounds
without a carrier has been shown to decrease infection rates in
clean orthopaedic and cardiac surgeries with infections
reduced from 1.8%–15% to 0%–2.5%, with doses ranging
from 0.25 to 2 g.9–13 When accounting for the cost of treating
SSIs, if it were to even make an improvement of infection rate
from 2.5% to 1%, the use of local antibiotic powder has been
estimated to save approximately $300 per case.9
Multiple animal models have evaluated this treatment,
one showing eradication when used immediately after
contamination and another showing significant decrease in
infection after establishing infection.14,15 The goal of this
study was to evaluate the effectiveness of local antibiotic
delivery in an established contaminated open fracture model
using doses comparable with those used in clinical practice.
We hypothesize that the application of intrawound antibiotics
will reduce bacterial contamination in this model.
MATERIALS AND METHODS
Study Design
The study was conducted in compliance with the
Animal Welfare Act, the implementing Animal Welfare
J Orthop Trauma  Volume 32, Number 10, October 2018
J Orthop Trauma  Volume 32, Number 10, October 2018
Regulations and in accordance with the principles of the
Guide for the Care of Use of Laboratory Animals. It followed
a protocol approved by the Institutional Animal Care and Use
Committee, and all care was performed under the supervision
of the Institutional Veterinarian. The model was based on
a previously described model that has been used to evaluate
multiple local wound adjuvants.16–20
Forty-five 400-g Sprague–Dawley rats were assigned
to 3 treatment groups: irrigation and debridement (I&D)
alone (control group, n = 15); I&D with placement of
PMMA beads containing vancomycin and tobramycin
powder (n = 15); and I&D with placement of intrawound
vancomycin powder (n = 15). All animals underwent iden-
tical procedures to create a contaminated fracture defect.
Animals were then treated 6 hours later with the prescribed
treatment for each group. Animals were killed 14 days later,
and samples were harvested and analyzed.
Bead and Antibiotic Preparation
Vancomycin hydrochloride hydrate powder was appor-
tioned into 10 mg aliquots for each rat, the equivalent of 2 g
of powder in an 80-kg human, a commonly used clinical
dose. For antibiotic beads, clinical treatment protocols were
replicated, using 40 g of Palacos R (Zimmer, Warsaw, IN)
powder mixed with 20 mL of methyl methacrylate monomer
and then 2 g of vancomycin and 2.4 g of tobramycin powder,
and then divided into 3-mm beads using a mold, each
containing 2.5 mg of vancomycin and 2.9 mg of tobramycin.
Procedure
Forty-five 400-g Sprague–Dawley rats (Charles River,
Wilmington, MA) were anesthetized with isoflurane 1%–2%,
a hind leg was shaved, prepped, and draped, and a standard
lateral approach to the femur was made. A sterile bespoke
polyoxymethylene plate (Special Designs, La Vernia, TX)
was fixated to the femur using 6 threaded Kirschner wires. A
6-mm defect was made in the midshaft of the femur using
a reciprocating saw. The defect was then inoculated with 30
mg of sterile bovine collagen (Sigma Aldrich, St. Louis, MO)
soaked with 1 · 105 colony-forming units (CFUs) of Staph-
ylococcus aureus (xenogeny strain) in 0.5 mL of saline. We
determined this dose, which produced consistent and repro-
ducible results, through several preliminary experiments
using variable doses of the test strain. The same dose was
used by Penn-Barwell et al,18 which was the most recent
previous study using this model. Wounds were closed in
layers, and animals were awakened and allowed to recover.
Six hours later, the animals were re-anesthetized, wounds
were reopened, and the designated treatment was performed.
In the control group, the wound was thoroughly debrided,
collagen was removed and was irrigated with 60 mL of sterile
saline using low pressure through a syringe. In the bead
group, the same was performed along with the addition of
the placement of 4 antibiotic beads containing 2.5 mg of
vancomycin and 2.9 mg tobramycin. In the powder group,
10 mg of vancomycin powder was spread throughout the
wound and rubbed into the tissues and the plate. Wounds
were closed in layers.
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Vancomycin and Contaminated Open Fracture Model
Rats were again allowed to recover, placed back into
their cages, and supplied with pain management drugs of
buprenorphine every 6 hours the first day then every 12 hours
daily for 3–7 days. They were allowed unrestricted activity in
their cages and monitored and given pain control. Fourteen
days later, they were given a lethal dose of carbon dioxide.
The femur and hardware were then removed. The disarticu-
lated femur was frozen in liquid nitrogen, crushed, and
homogenized with 10 mL of saline in an agitator. The plates
were first rinsed with 10 mL of phosphate-buffered saline
(PBS) to remove all loosely attached bacteria that is not part
of the biofilm. The plates were then placed in 2 mL of PBS
and vigorously vortexed to break the biofilm. The resus-
pended bacteria were serially diluted (10-fold) and 10-mL
aliquots were spotted onto nutrient plates to determine the
number of microorganisms (CFUs). To confirm that the bio-
film is completely removed from the plate, we removed the
vortexed plate, resuspended it in 2 mL of fresh PBS, and
vortexed again. The suspended material was again diluted
and plotted on nutrient plates. We recovered no CFU/mL
from the second vortexing, indicating the removal of the bio-
film completely during the first vortexing. All the samples
were sequentially diluted and spread on tryptic soy agar plates
and incubated overnight, and the bacterial colonies were
counted. A positive specimen was determined as 30 CFU/g
for the bone specimen or 30 CFU/mL for the plate specimen.
A photon count camera was used to verify bacterial strain.
Statistics
Fisher exact test was used to compare the presence or
absence of bacteria in samples. Mann–Whitney test was used
to compare quantitative cultures between groups for bone and
implant samples. Statistical significance was set at P , 0.05.
RESULTS
All samples from control group (15/15) and powder
group (15/15) showed bacterial growth in plates and bones
compared with 13 of 15 samples in the bead group (P = 0.48;
Fig. 1). Quantitative counts of bacteria in bone showed
FIGURE 1. Proportion of 15 samples from each treatment
group with detectable bacteria at 14 days. Editor’s Note: A
color image accompanies the online version of this article.
www.jorthotrauma.com | 539
Caroom et al
FIGURE 2. Quantitative bacterial counts from bone and plate
samples. Editor’s Note: A color image accompanies the online
version of this article.
significantly reduced growth in both bead expressed as (6.7 ·
105 CFU/g) [1.9 · 105 SEM] and powder (8.4 · 105) [1.4 ·
105] samples when compared with control samples (1.9 · 107)
[5.29 · 106] (P , 0.0001). Quantitative counts of bacteria in
plates showed significantly reduced growth in both bead (2.7 ·
105) [9.8 · 104] and powder (7.1 · 105) [2.1 · 105] samples
when compared with control samples (1.3 · 106) [2.4 · 105]
(P , 0.0003; 0.029). No significant differences were seen in
bacterial growth between bead and powder samples for either
bones (P = 0.13) or plates (P = 0.065) (Figs. 2, 3).
DISCUSSION
This study used a widely used contaminated open
fracture model and demonstrated that locally applied vanco-
mycin powder can be an effective adjunct in the treatment of
these injuries to decrease infection rates. This model has
showed decreased infection rates in the use intrawound
vancomycin powder similar to that of antibiotic beads, a long
established treatment adjuvant that is used in clinical settings.
FIGURE 3. Comparison of groups by quantitative cultures of
recovered bacteria from bone and implant samples (Mann–
Whitney test) and presence or absence of bacteria in samples
(Fisher exact test).
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J Orthop Trauma  Volume 32, Number 10, October 2018
Because the use of vancomycin powder does not require
a subsequent surgery for removal that comes with PMMA
bead placement, it certainly would reduce patient burden and
cost. The results did show a trend toward beads having better
quantitative results than powder; the presence of tobramycin
may have contributed, but this is largely due to 2 specimens
that demonstrated no growth, the only 2 in the study. Aside
from these, bacterial counts were quite similar.
The findings were similar to those seen in previously
conducted preclinical studies of intrawound vancomycin
powder; one with similar reduction in CFU in the same model
establishing infection with delayed treatment to simulate an
open fracture and one with eradication in a contaminated
surgical model where powder was applied immediately after
contamination.14,15 A study strength is the weight-based dosing
of the vancomycin powder applied to the wound. In an effort to
mimic dosing that has been applied in many clinical retrospec-
tive studies,9–13 10 mg was selected as the vancomycin dose
per wound, which is equivalent to 2 g in an 80-kg human. The
only other study that has evaluated intrawound vancomycin
powder in a rat model used 50 mg in 310 g rats, which is
equivalent to 12.4 g in an 80-kg human.15 This was selected
as the amount appropriate to coat the entire wound bed. Studies
of serum vancomycin levels when it is applied intrawound
show much lower systemic distribution than when given
intravenously.9,21
Further study might yield important information as to the
ideal dose of vancomycin powder when it is applied to a wound
bed. Other studies could investigate the magnitude of the effect
based on the dose or other factors that could be considered
such as a more rapidly bactericidal antibiotic or different
delivery method, possibly in liquid form to increase the area of
the wound coated. In addition to ongoing preclinical studies,
high-quality clinical trials are now underway with the Major
Extremity Trauma Research Consortium Vanco Trial,22 which
should give further insight into this treatment modality.
Study limitations include limitations inherent in any
animal model. Because of the surgical nature of the wound,
the local trauma to the soft tissues may not be as severe as that
seen in high-energy trauma wounds, so the devitalization of
local tissue may not be as severe as what is often seen
clinically. Additionally, we did not measure serum or tissue
levels of vancomycin after treatment. However, given the
lower levels that were used in comparison with other studies,
we felt levels would certainly be within acceptable ranges.
There are certainly concerns of the development of
vancomycin-resistant bacteria, but opinions differ on as to
the effect when applied locally, with some promoting the idea
that local application will decrease resistant organisms,9
whereas others worry that resistance could be promoted with
rapidly dropping tissue antibiotic levels.18
The novelty of this study is that it is the first to use doses
of vancomycin powder equivalent to that used clinically in
humans in an animal model in an established infection model.
Overall, this study suggests that the use of intrawound
vancomycin powder is an effective adjunct to irrigation and
debridement of contaminated open fractures and may be
equivalent to placement of antibiotic beads without requiring
subsequent removal.
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J Orthop Trauma  Volume 32, Number 10, October 2018
ACKNOWLEDGMENTS
The authors thank Tiffanie A. Brooks, DVM and the
Texas Tech University Health Sciences Center Laboratory
Animal Resources Center for their assistance with care and
management of the animal subjects and Nancy Swinford,
R.T.(R) (ARRT), CCRC, Clinical Research Coordinator
Texas Tech University Health Sciences Center Department
of Orthopaedics for project coordination.
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