Surgical Science, 2013, 4, 477-485
Published Online November 2013 (
Open Access SS
In Vitr o Mitigation of Pathogenic Bacteria and Virulence
Factors Using a Hydroconductive Dressing
Lauren T. Moffatt1, Rachel T. Ortiz1, Bonnie C. Carney1, Rachael M. Bullock1, Martin C. Robson2,
Marion H. Jordan1,3, Jeffrey W. Shupp1,3
1Firefighters’ Burn and Surgical Research Laboratory, MedStar Health Research Institute, Washington DC, USA
2Department of Surgery, University of South Florida, Tampa, USA
3The Burn Center, Department of Surgery, MedStar Washington Hospital Center, Washington DC, USA
Received October 7, 2013; revised October 31, 2013; accepted November 8, 2013
Copyright © 2013 Lauren T. Moffatt et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Wound infections can have devastating effects on healing as well as the health of the patient. Complications increase
when the pathogens are capable of producing virulence factors and/or are drug resistant. Novel methods are needed to
take on the challenges of treating such wounds. Drawtex® dressing is purported to have hydroconductive properties that
allow it to draw away debris and exudate from the wound into the dressing. The goal of this work is to better define
these interactions of this experimental dressing with bacteria and virulence factors. Two series of in vitro experiments
were performed. First, pieces of experimental dressing were submerged in a series of cultures in flasks and samples of
the dressing and cultures were taken over 90 minutes and assayed for bacteria and virulence factor levels. Second, ex-
perimental or standard care (control) dressings were placed on selective agar plated with pathogens of interest. Dress-
ings and the agar covered by them were used to quantify bacteria and virulence factors over time. The experimental
dressing took up both bacteria and virulence factors to a larger extent than the control dressing. Experimental dressing
significantly reduced the load of bacteria and virulence factors in cultures compared to control culture without dressing.
Based on the ability of the dressing to take up bacteria and virulence factors in this study, the data point to the potential
for this dressing to be similarly effective in reducing or eliminating pathogen from wounds, potentially increasing the
chances of successful wound healing.
Keywords: Infection; MRSA; Virulence Factors; Dressing
1. Introduction
Wound infections significantly increase the risk of pa-
tient morbidity and mortality [1,2], especially in in-
stances where the pathogens in question are able to pro-
duce exoproteins in the wound environment that increase
their virulence [3]. Many strains of pathogenic bacteria
commonly associated with wounds are capable of pro-
ducing an array of these virulence factors [4-8] (Table 1 ).
These factors such as hemolysins, collagenases, leuko-
cidins, and exotoxins can make the infection more inva-
sive, derail the local healing process, exacerbate the in-
flammatory response, and cause bacteremia and systemic
immune disruption [9-11]. Because of the incredible in-
fluence that virulence factors have on pathogenicity, re-
cent drug developments have included the oxazoladi-
nones and glycylcyclines, aimed via various mechanisms
at inhibiting protein synthesis and therefore abrogating
ability to produce virulence factors [12,13]. While this
has led to potential improvements in treating infections,
increasingly, strains of pathogenic bacteria observed in
Table 1. Wound-relevant bacteria species and associated vi-
rulence factors.
Species Virulence Factors
Toxic shock syndrome toxin 1 (TSST-1),
Panton-Valentine leukocidin (PVL), Staphylococcal
enterotoxins (SEB, SEA)
aeruginosa Exotoxin A, Phospholipase C
pneumoniae O-antigen, Endotoxin
baumannii OmpA, K1, Endotoxin
hospitalized patients as well as in outpatients, are multi-
drug resistant, presenting a new set of challenges in
Many topical agents and dressing products are aimed
at preventing infection by creating a barrier against con-
tamination, or have been designed to be bactericidal in
nature, often impregnated with antimicrobials such as
silver [14,15]. Drawtex® is a novel hydroconductive
dressing product that has been recently developed and is
reported to remove large quantities of exudate, bacteria,
and debris from wounds. In multiple case reports and
small clinical studies, it has been shown to decrease slough,
granulation tissue, and eschar in wound beds [16].
While multiple observations of the positive impact of
this dressing on healing potential have been documented,
little work has been done in a controlled model system to
systematically test its capabilities in mitigating bacteria
and virulence factor presence. Preliminary experiments
in controlled model systems that were aimed at deter-
mining the capabilities of Drawtex® in taking up bacteria
and protein from wounds and media were performed and
demonstrated that the dressing is in fact capable of mov-
ing significant amounts of both protein (albumin) and bac-
teria (Methicillin-resistant Staphylococcus aureus, MRSA)
from media and wounds [16]. The reduction of pathogen
and virulence factor load in wounds will impact the local
healing dynamic processes and immune response, poten-
tially removing the infectious complication and allowing
the progression of healing. If the dressing can achieve
this efficiently, it may succeed in advancing wound
healing and reducing pathogenicity of infection, where
antibiotics and topical bactericidal agents may fall short.
In order to further characterize the ability of Drawtex®
to interact with wound-relevant pathogens and their as-
sociated virulence factors, a series of in vitro experiments
were designed. Based on preliminary data, it was hy-
pothesized that the dressing will be able to effectively
decrease levels of bacteria and virulence factors in media.
Experiments were designed using liquid media in flasks
to demonstrate the absorption capabilities of the dressing
when saturated. A second set of experiments were de-
signed on solid agar media to more closely mimic a
“wound” surface and determine the ability of the dress-
ing to inhibit bacterial proliferation. The results de-
scribed here point to the potential for the experimental
dressing to serve a critical role in the management of
wounds as it may help mitigate invasive infections and
perhaps more importantly, reduce the pathogenicity of
the infection by reducing the presence of virulence fac-
2. Methods
2.1. Bacterial and Virulence Factor Absorption
To determine how effectively the experimental dressing
can take up wound-relevant bacteria species and their
associated virulence factors from media, three sterile
flasks containing 50 ml of Todd Hewitt (TH) Broth (BD
Biosciences, Franklin Lakes, NJ) were prepared. Two of
these flasks were then inoculated with methicillin-resis-
tant Staphylococcus aureus (MRSA) and incubated at
37˚C in a shaker overnight until the cultures contained
approximately 1 × 108 CFU/ml of MRSA. The strain of
MRSA used is a previously characterized clinical isolate,
known to produce multiple virulence factors. The third
flask served as a control, was not inoculated, and con-
tained 50 ml of TH broth only. Two pieces of the ex-
perimental dressing (Drawtex®) were cut to 3 cm × 3 cm,
with one piece submerged into one of the two MRSA
culture flasks and the other submerged in the uninocu-
lated TH broth (Figure 1). The second inoculated flask
was left without dressing.
The flasks were subsequently incubated at 37˚C with
gentle rocking for a total of 90 minutes, with samples of
submerged dressing and media from each flask collected
at 1, 10, 30, 45, 60, and 90 minutes post dressing sub-
mergence. Culture samples consisted of 0.5 ml aliquots
drawn from the flasks and then frozen at 80˚C, or used
immediately in quantitative cultures, while dressing sam-
ples were obtained using a 2 mm punch biopsy and were
weighed, flash frozen and stored at 80˚C. MRSA and
associated virulence factors of interest, specifically toxic
shock syndrome toxin 1 (TSST), Panton-Valentine leu-
kocidin (PVL), and alpha hemolysin (AH), were quanti-
fied in these samples using quantitative culture or ELISA
methods described below.
This experiment was performed in triplicate (n = 3)
and was then repeated using similarly prepared flasks
containing tryptic soy broth (Sigma-Aldrich, St. Louis,
MO) with approximately 1 × 108 CFU/ml of Pseudomo-
nas aeruginosa, Klebsiella pneumoniae, or Acinetobacter
baumannii. For each species, associated virulence factors
were quantified (Table 2). Statistically significant dif-
ferences between virulence factor and bacteria levels in
cultures absent of submerged dressing (controls) versus
levels in cultures containing dressing, were assessed us-
ing unpaired t-tests with a significance level of p < 0.05.
The third flasks containing uninoculated media and
dressing were assayed as described and no bacteria or
toxin were detected for any experiment at any time point
(data not presented). Data were plotted using GraphPad
Prism (GraphPad, La Jolla, CA, Version 6.02).
2.2. Bacterial and Virulence Factor Inhibition
To examine the impacts of the experimental dressing on
both bacterial growth and virulence factor production in
a controlled in vitro system, Staphylococcus aureus-se-
lective Mannitol salt agar (MSA) plates (BD Biosciences,
Franklin Lakes, NJ) were seeded with 100 µl of TH
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Figure 1. Absorption experimental design. Flasks prepared
with media, with or without bacterial inoculation and with
or without submerged experimental dressing, used in the
bacteria and virulence factor absorption experiments. Me-
dia and dressing samples from each flask were take n over a
time course with levels of bacteria and virulence factors
Table 2. Species and virulence factors studied in the present
Bacteria Species Studied Virulence Factors Examined
Pseudomonas aeruginosa Exotoxin A
Klebsiella pneumoniae Endotoxin
Acinetobacter baumannii Endotoxin
Broth containing 1 × 106 CFU/ml of MRSA. Experimen-
tal dressing (Drawtex®) or Mepilex® (Molnlycke, Nor-
cross, GA), used as a standard of care (SOC) control
dressing, were cut to 3 cm × 3 cm and placed in the cen-
ter of the inoculated MSA plates. Once daily from day 0
(placement of the dressing) to day 4, 2 mm punch biop-
sies of the dressings and the agar fields covered by them
(Figure 2) were collected, weighed, flash frozen, stored
at 80˚C and subsequently used to quantify bacterial
levels and virulence factors produced by the pathogen.
Digital photos of the plates were taken each day. The
experiment was performed in triplicate (n = 3). This ex-
periment was repeated using Leeds Acinetobacter (LA)
plates (Hardy Diagnostics, Santa Maria, CA) seeded with
Acinetobacter baumannii, MacConkey agar plates (BD
Biosciences, Franklin Lakes, NJ) seeded with Klebsiella
pneumoniae, and Pseuduomonas-selective agar plates
(Thermo Fisher Scientific, Waltham, MA) seeded with
Pseudomonas aeruginosa. Statistical analysis was per-
formed using unpaired t-tests (p < 0.05) to determine
differences in bacteria and toxin levels contained in the
Figure 2. Inhibition experimental design. Media plates used
for assessing bacteria and virulence factor inhibition of
experimental dressing, as compared to a standard of care
(SOC). Bacteria were plated and experimental or control
dressings were subsequently applied. Samples of the dress-
ings and the agar fields covered by them we re sampled over
a time course and bacteri a and virulence factors quantified.
experimental versus control dressings (and corresponding
agar fields covered by them) and data were plotted using
GraphPad Prism.
2.3. Quantitative Cultures
Dressing and agar samples were weighed, flash frozen in
liquid nitrogen and then stored at 80˚C. The flash fro-
zen samples were homogenized using a TissueLyser
(Qiagen, Germantown, MD), reconstituted with sterile
phosphate buffered saline (PBS), and vortexed. Culture
samples were assayed immediately after withdraw from
flasks. The homogenates or freshly obtained culture
samples were serially diluted with 100 µl of each dilution
plated on selective media plates. Plate selection de-
pended upon experimental bacteria; MSA plates selective
for S. aureus for MRSA quantification (yellow colonies
indicating coagulase positivity and presumptive S.
aureus), LA plates selective for Acinetobacter species
(light pink colonies, indicating alkaline products and
presumptive Acinetobacter), Pseudomonas-selective pla-
tes for Pseudomonas species (inhibition of growth of
other bacterial species), or MacConkey agar plates to
indicate Klebsiella species (pink colonies indicating lac-
tose fermentation and presumptive Klebsiella). Plates
were incubated at 37˚C for 24 hours, colonies of the ap-
propriate color were counted, and colony-forming units
(CFUs) per gram of agar or dressing, or per milliliter of
media were calculated.
In the absorption experiment (in flasks), fold changes
in CFU/g in dressing and CFU/ml in media over time
were calculated compared to data at t = 1 minute post-
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2.4. Virulence Factor Quantification at room temperature in the dark, and then 100 µl of 0.5%
of Sodium dodecyl sulphate (SDS) in distilled water
stopped the reaction. The plates were read at 405 nm in a
VICTOR Multilabel Counter (PerkinElmer, Waltham,
MA) and Workout 2.0 (PerkinElmer) was used to process
the results.
Enzyme-linked immunosorbent assays (ELISAs) were
used to quantify TSST-1, PVL, AH, and Pseudomonas
Exotoxin A (PEA). Dressing and agar samples were col-
lected, preserved and processed as described above with
homogenates reconstituted in PBS with 0.5% Tween 20
(PBST). Aliquots of 100 µl were added to wells of 96
well immunoassay plates (Nalge Nunc International,
Rochester, NY) coated with 100 µl of a 1 mg/ml primary
antibody raised to TSST-1, AH (ToxinTechnology, Inc.,
Sarasota, FL), PVL (Integrated Biotherapeutics, Gaither-
sburg, MD), or PEA (Sigma-Aldrich, St. Louis, MO).
Culture samples and a serial dilution of standard purified
TSST-1, PVL, AH, or PEA were treated in the same way.
The plates containing samples and standard curve were
then incubated at 37˚C for 2 hours, washed with PBST,
and then 100 µl of secondary antibody diluted 1:300 in
PBST was added to each well. The plates were placed on
a shaker and incubated at 37˚C for 1 hour and then
washed with PBST. Each well then received 100 µl of
2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS)
with 0.05 M phosphate citrate buffer (Sigma-Aldrich) and
hydrogen peroxide. The plates were sealed and incubated
A. baumannii endotoxin (AE) and K. pneumonia en -
dotoxin (KE) were both quantified in culture samples and
dressing and agar samples (processed as above) using a
Pierce LAL Chromogenic Endotoxin Quantitation Kit
(Thermo Fisher Scientific, Rockford, IL).
3. Results
3.1. Bacterial and Virulence Factor Absorption
Bacteria were not detected in any of the baseline dressing
samples (pre-submergence), or in the uninoculated TH
broth throughout the time course. Starting at 10 minutes
post-dressing submergence, all cultures that contained
submerged experimental dressing had a significantly
lower bacterial count compared to the control culture
without dressing (Figure 3). A decreasing trend in bacteria
levels in the cultures containing dressing continued over the
Figure 3. Fold change from bacteria levels quantified at 1 minute post-dressing submergence in cultures or dressing samples
over time. Comparisons in bacteria levels between cultures containing experimental dressing versus cultures without dressing
were made at each time point using an unpaired t-test (p < 0.05) with statistically significant difference indicated (*).
90 minute time course, while at the same time, bacteria
quantified in the dressing samples increased correspond-
ingly (Fi gure 3).
TSST1 levels in the MRSA culture containing experi-
mental dressing were significantly lower compared to the
control culture (without dressing) by 30 minutes post-
dressing submergence (Figure 4(a)). At 45 minutes into
the time course, AH, PVL, KE, and AE levels quantified
in cultures of associated bacteria containing the dressing
were significantly lower compared to the control cultures
(Figures 4(b)-(f) respectively). By 60 minutes, PEA was
also significantly lower in dressing-containing Pseudo-
monas cultures compared to the control. Similar to the
trends in bacterial levels in the dressing submerged in
cultures, virulence factor levels also showed an increase
over time, as levels in the media decreased (Figure 4).
3.2. Bacterial and Virulence Factor Inhibition
Upon examination of the inoculated plates for all species
of pathogen studied, noticeable differences existed be-
ginning 24 hours after placement of the dressings on the
inoculated plates (study day 1) in the number of colonies
in agar fields covered with SOC control dressing com-
pared to experimental dressing-covered fields (digital
images not shown). Beginning on day 1 of the experi-
ment, this difference was statistically significant in plates
seeded with MRSA and P. aeruginosa (Figures 5(a) and
(b)), while the difference in bacteria quantified in ex-
perimental fields was significantly lower than that in
control fields by day 2 for K. pneumoniae and A.
baumannii (Figures 5(c) and (d)). Decreasing trends in
bacteria continued through day 4 and was similar for all
Figure 4. Levels of TSST1 (a), AH (b), PVL (c), PEA (d), KE (e), or AE (f) quantified in culture or dressing samples over a 90
minute time course in the absorption experiments. Statistically significant (*) differences in levels in culture samples contain-
ing dressing as compared to cultures without dressing were determined at each time point using an unpaired t-test (p < 0.05).
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Figure 5. Colony forming units of bacteria per gram (CFU/g) of inoculated agar samples covered with either experimental or
control dressings, quantified over 4 days. Statistically significant differences (*) in bacteria levels between experimental
dressing-covered agar and control dressing (SOC)-covered agar samples at each time point were assessed using an unpaired
t-test (p < 0.05).
species of pathogen examined (Figure 5). While the agar
fields covered with experimental dressing had lower lev-
els of bacteria than those covered with control dressing,
at the same time there was a significantly higher level of
bacteria quantified in the experimental dressing com-
pared to the control dressing at all time points tested and
for all species (Figure 6).
Virulence factor levels were also reduced in the agar
covered by experimental dressing compared to the con-
trol dressing-covered agar. Some virulence factors
(TSST1, PVL, and PEA) became non-detectable in ex-
perimental agar fields early in the time course (Figures
7(a), (c) and (d)) while control dressings were not capa-
ble of reducing any of the factors below detection limits
within the four day experiment (Figure 7). For those
virulence factors not already below detection limits in
experimental agar fields, levels were significantly lower
as compared to controls by day 2 (AE) or 3 (AH and KE)
(Figures 7(b), (e) and (f) respectively). As patterned in
the bacteria data, virulence factor levels showed a trend
of increase in the experimental dressing over the time
course, as levels in the agar fields decreased (Figure 8).
Experimental dressing had significantly higher levels of
TSST1 and PVL than the control dressing by study day 1
(Figures 8(a) and (c)) and this was also true for levels of
PEA (Figure 8(d)). Levels of AE quantified in experi-
mental dressing were significantly greater than those in
control dressings by day 2 (Figure 8(f)), while AH and
KE levels were significantly higher by day 3 (Figures
8(b) and (e)).
4. Discussion
The first set of experiments described here was designed
to examine the ability of the experimental dressing to
take up both bacteria and virulence factors from bacterial
culture. A consistently lower level of both bacteria and
virulence factors was quantified in the cultures that con-
taied submerged experimental dressing when com- n
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Figure 6. Colony forming units of bacteria per gram (CFU/g) of either experimental or SOC control dressing after 1, 2, 3, or 4
days of covering an agar field inoculated with bacteria. Statistically significant differences (*) in bacteria levels between ex-
perimental dressing and control dressing samples at each time point were assessed using an unpaired t-test (p < 0.05).
Figure 7. Levels of TSST1 (a), AH (b), PVL (c), PEA (d), KE (e), or AE (f) quantified in inoculated agar samples covered with
either experimental or control dressings, quantified over 4 days. Statistically significant (*) differences in levels in control
samples as compared to experimental samples were determined at each time point using an unpaired t-test (p < 0.05).
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Figure 8. Levels of TSST1 (a), AH (b), PVL (c), PEA (d), KE (e), or AE (f) quantified in experimental or SOC control dress-
ing after 1, 2, 3, or 4 days of covering an agar field inoculated with bacteria. Statistically significant (*) differences in levels in
control dressing as compared to experimental dressing were determined at each time point using an unpaired t-test (p < 0.05).
pared to the control cultures that did not contain any
dressing (Figure 3). At the same time, there was a con-
sistent increase over the time course in bacteria and viru-
lence factors quantified in the dressing itself throughout
the study (Figures 3 and 4). This demonstrates a clear
capability to absorb both gram positive and gram nega-
tive pathogens and a variety of proteinaceous virulence
factors, indicating a potential role in removing the same
contaminants from wound exudate and fluids. Moreover,
this hints at interesting mechanisms of action that allow
the dressing material to accumulate materials with di-
verse structural and biochemical properties over time.
The second set of experiments described demonstrated
a significant mitigation of gram positive and gram nega-
tive bacterial presence, as well as reduced virulence fac-
tor levels, in plates seeded with pathogens of interest
(Figures 5 and 7). Compared to an SOC control dressing,
the experimental dressing appeared able to accumulate
pathogen and protein of interest to a larger extent (Fig-
ures 6 and 8), and in turn, eliminate both to a higher de-
gree from the agar. As these data show, over a period of
days the dressing is capable of steadily accumulating
additional pathogen and virulence factors. If the experi-
ment had been taken out for a longer time period, it
would be hypothesized that the dressing may reach a
“saturation” point, perhaps dependent on the bacterial
load present on the media. Whether this would occur
before all detectable bacteria or toxin was eliminated
from the media, it is a reasonable question, and is likely
dependent on the amount of pathogen and virulence fac-
tor present when the dressing is applied. If in fact a
maximum capacity exists for the dressing, further ques-
tions would include whether the contaminants taken up
by the dressing would eventually then be released back
into the agar; or rather, how long they can be retained.
Further experiments should be done to evaluate this, as
clinical implications may exist related to frequency of
dressing changes and whether the presence of contami-
nants in the dressing would, over some amount of time,
impact the wound being treated.
It should also be noted that while Drawtex® showed
much more significant impacts on pathogen and viru-
lence factor mitigation in the plated agar models, the
SOC control did demonstrate similar trends of decrease
in bacteria and pathogen in the fields covered, though to
a much more minimal extent and not for all of the bacte-
ria and virulence factors examined.
The goal of the work described here was to character-
ize the efficacy of this experimental dressing in abrogat-
ing multiple types of bacteria and the associated viru-
lence factors in vitro. To date, no research has been done
to characterize the interactions of this new dressing with
common wound pathogens, and the protein products that
result in increased pathogenicity. This work should serve
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as a foundation for further controlled studies, both in
vitro and in vivo, to fully understand the capabilities of
this promising dressing in treating wounds.
5. Acknowledgements
This work was funded in part by SteadMed Medical and
by the DC Firefighters’ Burn Foundation.
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