David Parsons1 PhD, MRSC CChem Akhil Seth2 MD, BS Thomas Mustoe2 MD, FACS 1.ConvaTec Research & Development, Flintshire, Wales, UK 2.Division of Plastic Surgery, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

1. David Parsons1 PhD, MRSC CChem
Akhil Seth2 MD, BS
Thomas Mustoe2 MD, FACS
1. ConvaTec Research & Development, Flintshire, Wales, UK
2. Division of Plastic Surgery, Feinberg School of Medicine,
Northwestern University, Chicago, Illinois, USA.
1EWMA 2013 Poster EP563
EP563
A new anti-biofilm dressing:
in vivo investigation
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2. A new anti-biofilm dressing: in vivo investigation
ABSTRACT
Aim: To use an in vivo wound biofilm model to quantify the effect of a new absorbent enhanced-antimicrobial silver
dressing (EASH) on reducing bioburden and improving wound healing rates.
Methods: As described by Gurjala1, biofilms of a wild-type strain of Pseudomonas aeruginosa (PA01) were grown in
a well characterized and validated full-thickness wound model in the rabbit ear. The study had three arms: i)
wounds were treated with EASH, ii) EASH without the antimicrobial components (Non-EASH), and iii) a non-adherent
gauze dressing containing PHMB (PHMB gauze). Dressings were changed every alternate day and quantitative
bacterial counts were performed at each change. After six days of treatment the animals were euthanized, wounds
excised and healing assessed by quantitative microscopy.
Results: EASH reduced biofilm bioburden more rapidly (~57% per day, statistical significance) than either Non-EASH
or PHMB gauze (~17% per day). A statistically significant difference was found for wound healing as measured by
granulation and epithelial gaps (respectively, 10% and 19% smaller with EASH than with PHMB gauze) and
granulation tissue formation and epithelialization areas (48% and 24% larger with EASH than with PHMB gauze).
Conclusion: Even in acute wounds in healthy animals, the presence of biofilm delays healing. EASH is more effective
in reducing biofilm, and thus restoring normal acute-wound healing rates, than either the base dressing (non-EASH)
or the PHMB gauze.
1. Gurjala et al (2011). Wound Repair Regen. 19, 400-410.
EWMA 2013 Poster EP563

3. EWMA 2013 Poster EP563 3
“A growing body of evidence suggests that in addition to hypoxia, ischemia-reperfusion injury, and intrinsic host
factors, bacterial biofilms represent a fourth major pillar in chronic wound pathogenesis.” 1
To better understand the behaviour and clinical consequences of biofilms and how they may be effectively treated,
controlled studies are required. Human studies are logistically and ethically prohibitive and they are also impossible
to standardize. In vitro models have been developed but these are not always representative and are unable to
provide concurrent information about wound healing.
The research group at Northwestern University has adapted a rabbit dermal ulcer model (an FDA-recognized model
of wound healing in use for 20+ years) to give a reproducible biofilm-containing wound that is sub-infection1.
The following have been demonstrated:-
- Consistent (healing observations, bacterial counts)
- Control wounds heal like human wounds
- The presence of wound biofilm (by scanning electron microscopy (SEM))
- Quantitative & qualitative analyses at multiple time-points
1. Gurjala et al (2011). Wound Repair Regen. 19, 400-410.
Introduction
Control wounds Biofilm-colonized wounds Infected wounds
biofilm purulence & debris
Biofilm-colonized wounds do not exhibit the
purulence and debris associated with actively-
infected wounds; rather ‘films’ of bacteria are
observed across the wound

4. Rabbit ear model for studying biofilm & healing
1. 6-mm-diameter full-thickness wounds made in ears of New Zealand white rabbits
2. Wounds inoculated with 1,000,000 (106) c.f.u. challenge microorganism(s)
3. Covered with a polyurethane film dressing† to allow biofilm formation over 6 days
4. Apply topical antibiotics (& optionally PHMB gauze‡) to eliminate planktonic bacteria, maintaining biofilm
colonization
Using this model it has been shown that:-
• Biofilm causes lower-grade inflammatory response than active infection1,2; two-species biofilm elicits greater
inflammatory response than single-species3
• Biofilm significantly inhibits granulation and epithelialization (the gap is wider and area smaller), therefore
healing rate is reduced1,2,3,4
• Different species of biofilm impair healing to different extents (P. aeruginosa > S. aureus > K. pneumoniae)4
1. Gurjala et al (2011). Wound Repair Regen. 19, 400-410.
2. Seth et al (2012). Wound Repair Regen. 20, 214-224.
3. Seth et al (2012). PloS ONE, 7, e42897.
4. Seth et al (2012). J. Am . Coll. Surg. 215, 388-399. 4EWMA 2013 Poster EP563
† TegadermTM
‡ TelfaTM AMD
Host inflammatory cells Biofilm
HistologyElectron microscopy
EG = epithelial gap

5. EWMA 2013 Poster EP563 5
To use this in vivo wound biofilm model to quantify the effect of a new absorbent enhanced-antimicrobial silver
dressing (EASH)† on reducing bioburden and improving wound healing rates
Pseudomonas aeruginosa (wild-type strain PA01) biofilm-colonized wounds were created over a 6-day period
Wounds were treated for 6 days, with dressing changes every other day:
- Half the wounds on each animal were covered with PHMB-containing gauze#
- The other half were covered with either EASH† or the non-antimicrobial vehicle dressing‡
At each dressing change: clinical observations were made and samples were taken for microbiological analysis
On sixth treatment day: histological samples were taken to measure new (and missing) granulation tissue and
epithelium
Aim
† AQUACEL  Ag+ dressing (EASH)
‡ AQUACEL dressing (non-antimicrobial vehicle)
# TelfaTM AMD dressing (PHMB-containing gauze)
Method

6. Results – bioburden reduction
EASH significantly reduced bioburden of biofilm-colonized wounds compared to the non-antimicrobial vehicle and
PHMB-containing gauze dressings:
- ~57% reduction in biofilm bioburden per treatment day with EASH dressing *
- Only ~17% reduction per treatment day with non-antimicrobial vehicle or PHMB-containing gauze
6EWMA 2013 Poster EP563
10.000
100.000
1.000.000
10.000.000
100.000.000
2 4 6
Meanviablebacterialcount(cfu/wound)
(n=6)
Days of Treatment
PHMB-containing Gauze
Non-antimicrobial Vehicle
EASH
* p < 0.05
*
*
*
*

7. EASH significantly improved the rate of granulation tissue formation in biofilm-colonized wounds compared to the
non-antimicrobial vehicle and PHMB-containing gauze dressings:
- 10% smaller granulation gap with EASH compared to PHMB-containing gauze *
- 48% larger granulation area with EASH compared to PHMB-containing gauze *
Results – granulation tissue formation
7EWMA 2013 Poster EP563
0
1
2
3
4
5
PHMB-containing
Gauze
Non-antimicrobial
Vehicle
EASH
GranulationGap(mm)
(n=18)
Granulation Gap
*
*
0
20
40
60
80
100
120
140
PHMB-containing
Gauze
Non-antimicrobial
Vehicle
EASH
GranulationArea(mm2)
(n=18)
Granulation Area *
*
* p < 0.05

8. Results – epithelialization
EASH significantly improved the rate of epithelialization of biofilm-colonized wounds compared to the non-
antimicrobial vehicle and PHMB-containing gauze dressings:
- 19% smaller* epithelial gap with EASH compared to PHMB-containing gauze
- 24% larger* epithelial area with EASH compared to PHMB-containing gauze
8EWMA 2013 Poster EP563
0
1
2
3
4
5
PHMB-containing
Gauze
Non-antimicrobial
Vehicle
EASH
EpithelialGap(mm)
(n=18)
Epithelial Gap
*
*
0
20
40
60
80
100
120
PHMB-containing
Gauze
Non-antimicrobial
Vehicle
EASH
EpithelialArea(mm2)
(n=18)
Epithelial Area
*
*
* p < 0.05

9. Conclusion
• Controlled animal models can be used to study the biological consequences of wound
biofilm
• Even in acute wounds in healthy animals, the presence of biofilm delays healing
• Compared to a non-antimicrobial vehicle dressing and a PHMB-containing gauze
dressing, a new absorbent enhanced-antimicrobial silver dressing (EASH) was shown to:-
 Have a significantly greater reduction in wound bioburden (biofilm)
 Significantly improve the rate of granulation tissue formation
 Significantly improve epithelialization rate
9EWMA 2013 Poster EP563