EWMA 2013 - Ep563 - A new anti-biofilm dressing: in vivo investigation

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 EWMA 2013 Poster EP563

EP563

A new anti-biofilm dressing: in vivo investigation

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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

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


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

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


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.


† TegadermTM ‡ TelfaTM AMD

Host inflammatory cells Biofilm

Histology Electron microscopy

EG = epithelial gap

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

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


10.000

100.000

1.000.000

10.000.000

100.000.000

2 4 6

Me

an v

iab

le b

acte

rial

co

un

t (c

fu/w

ou

nd

)

(n=6

)

Days of Treatment

PHMB-containing Gauze

Non-antimicrobial Vehicle

EASH

* p < 0.05

* *

* *

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


0

1

2

3

4

5

PHMB-containingGauze

Non-antimicrobialVehicle

EASH

Gra

nu

lati

on

Gap

(m

m)

(n=1

8)

Granulation Gap

* *

0

20

40

60

80

100

120

140



EASH

Gra

nu

lati

on

Are

a (m

m2)

(n=1

8)

Granulation Area * *

* p < 0.05

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


0

1

2

3

4

5



EASH

Epit

hel

ial G

ap (

mm

) (n

=18

)

Epithelial Gap

* *

0

20

40

60

80

100

120



EASH

Epit

hel

ial A

rea

(mm

2)

(n=1

8)

Epithelial Area

* *

* p < 0.05

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


EWMA 2013 - Ep563 - A new anti-biofilm dressing: in vivo investigation

Health & Medicine

Transcript of EWMA 2013 - Ep563 - A new anti-biofilm dressing: in vivo investigation