1

An in vitro study of the antimicrobial activity and efficacy of iodine-generating

hydrogel wound dressings

R.M.S. THORN BSc*, J. GREENMAN BSc PhD** AND A. AUSTIN BSc+

*Research Assistant, Department of Microbiology, University of the West of England,

UK

**Professor of Microbiology, Department of Microbiology, University of the West of

England, UK

+Development Scientist, Insense Ltd., Bedford, UK

To whom all correspondence should be addressed:

Professor John Greenman

Faculty of Applied Sciences, University of the West of England, Frenchay Campus,

Coldharbour Lane, Bristol, BS16 1QY, United Kingdom

Telephone: 0117 344 2515

Fax: 0117 344 2904

E-mail: John.Greenman@uwe.ac.uk

2

Abstract

Objectives: To determine the antimicrobial activity and efficacy of different

formulations of novel bioxygenating hydrogels against various target organisms by

means of an in-vitro test system that more effectively mimics the conditions

encountered when dressings are in contact with wounds.

Methods: Cellulose filter discs (n = 32) were inoculated with indicator species and

placed equidistant from each other as a matrix onto agar test beds. Cut squares of

control or test dressings were placed on top of each disc. Kill curves were constructed

from determinations of the numbers of survivors (log cfu disc-1

) over time by

removing disc samples at various time points.

Results: Significant differences (p<0.05) were observed between controls and test

samples. The order of sensitivity for the oxyzyme hydrogel was Fusobacterium

nucleatum, Bacteroides fragilis, Propionibacterium acnes, Staphylococcus

epidermidis, Staphylococcus aureus (MRSA), Candida albicans and Pseudomonas

aeruginosa. The order of efficacy of the hydrogel dressing formulations (iodozyme

402>iodozyme 401>oxyzyme) was the same regardless of the target species.

Conclusions: The novel hydrogel skin surface wound dressings are broad spectrum in

activity, encompassing antibiotic resistant organisms, anaerobes and yeasts, and the

antimicrobial function appears to be rapidly effective.

3

Introduction

Numerous commercial antimicrobial wound dressings are already available, most of

which are based on silver as the active ingredient, and new versions are constantly

being introduced onto the market. Practitioners and other decision-makers are,

therefore, faced with a need to make rational choices between different supposedly

antimicrobial dressings, but there is little objective evidence on which to base these

choices. There is a real need to understand more about the complex underlying nature

of wound infections, as well as the interaction between the wound, the microbial flora

and the dressing. Simple laboratory evaluations of antimicrobial potency can be very

misleading, especially with modern composite dressings, because the tests fail to take

account of this complex interaction. For these reasons, there is a particular need for

in-vitro laboratory tests that take into account at least the most basic aspects this

interplay of factors, especially the way in which wound microbes are bathed in fluid

rich on organic, nutritional substances, most of which are drawn into the dressing

where they can inactivate antimicrobial agents.

Wound management must always take into account the risk of clinical infection

becoming established. Microbial colonisation occurs in virtually all acute, traumatic,

surgical and chronic wounds. Often, colonisation involves potentially pathogenic

organisms that can lead to a wound becoming infected with organisms that cause

serious complications. It is widely recognised that such infections delay healing,

thereby causing increased trauma to the patient and increasing treatment costs, which

isa the reason for a growing demand for effective wound management and therapeutic

options to limit the risk of infection1. Burns, diabetic foot ulcers, leg ulcers and

4

decubitus ulcers are particularly prone to complications resulting from infection. For

example it is estimated 75% of deaths following burn injuries are related to infection2.

Although systemic antibiotics are regarded as the ultimate treatment against

wound infection, antiseptic skin surface dressings have long been used to control

wound micro flora and, in normal clinical practice, to combat infection. Antibiotics

have been shown to be inappropriate in some cases3, and can result in wound

colonization by resistant organisms4. When treating wounds colonised with bacteria

resistant to conventional antibiotics (e.g. MRSA), it is important to contain the

bacteria within the colonised wound to prevent cross-infection (particularly in a

hospital environment), thus making wound management through skin dressings

essential.

There is wide variation in the efficacy of currently available skin surface

dressings at combating wound infection. It has been shown that the rate of clinical

infection is lower under occlusive dressings than non-occlusive dressings5 and that, in

contrast to dry dressings (e.g. absorbent cellulose), dressings that promote moist

wound healing (e.g. hydrocolloids) offer external protection to the wound and limit

the spread of bacteria by aerosol formation upon removal6. More recently Bowler et

al7 demonstrated the effective sequestering and retention of micro-organisms by a

hydrofiber dressing in vitro, which could help to reduce the microbial load in wounds

and the surrounding environment.

Antimicrobial agents can be incorporated into skin surface wound dressings,

although this tends to be limited to antiseptics, as the routine use of topical antibiotics

to treat colonized or infected wounds is seen as unjustified8. Although the use of

antiseptics in wound care has long been debated, a recent wide-ranging review of

published data by Drosou et al9 concluded that “antiseptics need not be omitted from

5

the therapeutic armamentarium of wound care”, quoting limited toxicity data, broader

antimicrobial spectrum and lower sensitization rates. Because antiseptics incorporated

into skin surface wound dressings are in contact with the wound for much longer than

in solution form, they can be more dilute, less toxic, and exert a prolonged

antimicrobial effect8.

As wound dressing technology becomes more advanced, new developments

are emerging in which antimicrobial agents are delivered in a controlled manner from

composite dressings that also interact with the wound. For example, the new

OXYZYME (oxyzyme) and IODOZYME (iodozyme) wound dressings utilise an

enzyme (glucose oxidase) to drive the transport of oxygen into the wound and the

synthesis in-place of a predetermined dose profile of iodine (fig 1.). The integration

of antiseptic iodine with oxygenation of the wound bed provides an effect described

as wound “bioxygenation”. Currently, these biochemically active wound dressings

are built into a hydrogel matrix, consisting of three dimensional networks of

hydrophilic polymers that are flexible, non-antigenic, and capable of absorbing large

amounts of wound fluid, as well as providing a barrier to external infection10

. A moist

wound healing environment is created by the occlusive nature of the hydrogel sheets,

as well as their ability to donate moisture. This moist environment has been shown to

be associated with decreased healing time, greater patient comfort and reduced

costs11

.

The steady release of iodine exerts a gentle, surface antimicrobial effect at the

interface between wound and dressing, ideal in treatment of wounds where the fear of

infection is an issue. This antimicrobial effect can be further enhanced by adjusting

the reaction conditions within the gel layers to create the iodozyme system, in which

higher levels of iodine are synthesised. In principle, the system allows for any

6

practicable level of iodine to be generated, simply by appropriately modifying

concentrations of enzyme and iodide, as well as pH, surfactant concentration and the

dimensions of the gel layers.

The antimicrobial activity of such composite, multifunctional dressings is not

easily evaluated by any of the previously established in-vitro methods, since it is

essential for any such evaluation to take account of the system’s controlled

mechanism of action and its potential to interact with the wound. The key features of

oxyzyme and iodozyme hydrogel systems to be taken into account are the mechanism

and dynamics of iodine generation, the associated oxygen production and the steady

take-up of wound-fluid (which causes an associated swelling and dilution of the

biochemical constituents). Under aerobic, in-vitro conditions, it is difficult to

measure the full impact of the oxygenation effect (which depends on the involvement

of leukocytes), even though the oxygen delivery mechanism will be active. However,

the new test system used in this study enables the antimicrobial efficacy of the gradual

synthesis and release of iodine from the dressings to be determined. Moreover the test

system also allows evaluation against anaerobes.

The specific aim of this in vitro study was to determine the antimicrobial

activity and efficacy of three variants of this novel bioxygenating hydrogel system.

However, the general approach behind the the test system is more widely useful for

the comparison and evaluation of any dressing claimed to have antimicrobial effects.

The general test system utilised here, can be used help make rational decisions as to

which active/advanced dressing to use in wound management, especially as it is

important to objectively determine antimicrobial activity against a broad spectrum of

target species, including MRSA, anaerobes and yeasts. The efficacy of dressings

7

against anaerobes is often over-looked, despite the high correlation between the

incidence of anaerobic bacteria and wound infection1.

Materials and methods

Growth and maintenance of micro-organisms

Within this study a mixture of clinical and laboratory typed strains were used to

enable both comparisons with existing data for other novel antimicrobial dressings,

and determination of antimicrobial efficacy against clinically relevant pathogens.

Staphylococcus epidermidis (UWE laboratory typed strain isolated from human skin),

Methicillin-Resistant Staphylococcus aureus (MRSA) Llewelyn clinical strain,

Pseudomonas aeruginosa PAO 1161, Candida albicans NCTC 10288,

Propionibacterium acnes (UWE laboratory typed strain isolated from human skin),

Bacteroides fragilis ATCC 25285 and Fusobacterium nucleatum (subspecies

nucleatum) ATCC 10953 were all maintained and stored as laboratory stocks.

Staphylococci and Pseudomonas were maintained on Nutrient Agar (Oxoid Ltd,

Basingstoke, UK), C. albicans on Potato Dextrose Agar (Oxoid Ltd, Basingstoke,

UK), and P. acnes and the strict anaerobes on Fastidious Anaerobe Agar (Oxoid Ltd,

Basingstoke, UK) supplemented with 5% defibrinated horse blood (TCS Biosciences,

Buckingham, UK). Broth cultures of S. epidermidis, S. aureus (MRSA), Ps.

aeruginosa, C. albicans and P. acnes were grown in 1% tryptone-0.5% yeast extract,

and B. fragilis and F. nucleatum in brain-heart infusion broth. The staphylococci,

Pseudomonas and Candida were incubated at 37ºC aerobically (Genlab M1005L

incubator, Cheshire, UK), and P. acnes and strict anaerobes were incubated at 37ºC

anaerobically (MK3 Anaerobic work station, Don Whitley Scientific, Shipley, UK).

All cultures were sub-cultured weekly.

8

Experimental wound dressing

The wound dressings tested were prototype oxyzyme and iodozyme hydrogels

(Insense, Bedford, UK). The oxyzyme hydrogels comprised a 100 x 100mm glucose +

iodide wound contact layer, and a 60 x 60mm enzyme surface layer. The enzyme

layer was responsible for the hydrogen peroxide driven oxygen transport and iodine

generation from the wound contact layer (fig 1.). The essentially similar iodozyme

hydrogels were supplied at two iodide concentrations (Test Hydrogels 401 & 402),

again comprising a 100 x 100mm glucose + iodide wound contact layer and a 100 x

100mm enzyme surface layer. These generated and released higher concentrations of

iodine. The concentration of iodide in hydrogel 402 was twice that of hydrogel 401.

All wound dressings were supplied in their standard packaging, and cut into 16

sections of required size just prior to use.

Disc inoculation

Test cell suspensions were prepared from neat 24hr broth cultures of staphylococci

and Ps. aeruginosa, 48hr broth cultures of C. albicans and 72hr broth cultures of P.

acnes and strict anaerobes, diluted if necessary with sterile broth to give optical

density readings between 0.5 and 0.7 using a 1cm light path CE-373 linear readout

grating spectrophotometer (Cecil Instruments, Cambridge, UK) at wavelength 540nm.

UV-sterilised 1cm2 cellulose discs (n = 32) were then immersed in vortex mixed test

cell suspensions for 5 minutes, removed into a sterile plastic petri-dish and excess

fluid removed from each disc by sterile blotting. All discs then contained

approximately the same numbers of viable cells12

.

Test Method

The test method used was that described previously by Thorn et al.12

(Fig 2.). Briefly,

test and control wound dressings (n = 16, 25 x 25mm) were evenly distributed on to

9

the surface of 10mm thick 1% w/v agar in 1% w/v tryptone-0.5% w/v yeast extract

agar within a large square assay plate. Test dressing samples comprised of the iodide

plus glucose wound contact layer and the enzyme surface layer, whereas the control

wound dressing (control 1) comprised of only the iodide plus glucose wound contact

layer without the surface enzyme layer (preventing hydrogen peroxide and iodine

generation) (fig 2.). Sample positions were determined by reference to a grid matrix

(intersection of rows and columns) and the sequence of samples determined according

to latin square principles. The test plates were pre-incubated for two hours (to

equilibrate) prior to the introduction of the test disks containing equivalent numbers of

target species. These were placed between the agar layer and the test and control

dressings using sterile forceps. Small metal weights (12 g) on nylon mesh pads were

then applied onto the top surface of each hydrogel square in order maintain contact

between hydrogel, cellulose disk and agar surface during incubation. Uncovered disks

(n=16) were also placed onto the test plates containing equivalent numbers of target

species as an additional uncovered control (control 2). All assay plates were incubated

at 25°C for the duration of the experiments.

Sample discs were removed at appropriate times (determined from preliminary

experiments to roughly estimate kill rates) and the numbers of survivors determined

by viable count as previously described12

.

Analysis of results

For each test species and each condition, plots were made of log numbers of survivors

over reaction time and kill rates determined by linear regression, enabling direct

comparison of the results over time for the test and two controls for each target

species. Graph construction, statistical analyses, and modelling were conducted with

the use of GraphPad Prism version 3.02 for Windows (GraphPad Software, San

10

Diego, CA, USA). An Analysis of Covariance (ANCOVA) was used to ascertain

whether there was a significant difference between rates. A P value of <0.05 was

regarded as significant. In order to take into account any change in cfu per cm-2

within

control samples and to allow direct comparison of all test organisms and conditions,

log plots were also constructed using the following formula13

:

Y = (log Nt/No)test − (log Nt/N0)control

Where: No = cfu cm-2

at zero time and Nt = cfu cm-2

at later time (t)

Results

All hydrogel wound dressings exhibited a significant antimicrobial effect against all

test organisms. Plotting log numbers of survivors over the reaction time enabled the

determination of kill rates (table 1). The death kinetics for S. epidermidis12

and S.

aureus MRSA (fig 3.) treated with the oxyzyme dressing exhibited an accelerating

kill rate. For the purposes of comparison with other species and conditions, an

average kill rate value was estimated between the maximum and minimal rates of the

acceleration. All test kill rates were significantly different (p<0.0001) than either no

change (flat line) or either of the two corresponding control conditions, and one

another.

When the low iodine oxyzyme hydrogel was tested against Ps. aeruginosa

there was a significant antimicrobial affect for the first 6½ hours of treatment (fig 4.).

However after this period significant re-growth occurred, eventually reaching levels

close to that of both controls, even though both sets of control samples exhibited

significant growth rates. No such re-growth was seen during treatment with iodozyme

hydrogels. Significant growth of control samples was also observed for C. albicans,

11

however all test hydrogels exhibited a significant kill rate throughout the duration of

treatment (fig 5.).

For all anaerobes (P. acnes, B. fragilis and F. nucleatum) treated with the

oxyzyme hydrogel, significant kill rates were observed for both test and control

samples (figs 6-8.). However, test conditions (in the presence of active hydrogels)

gave the highest kill rates and were significantly different from control kill rates

(p<0.0001).

Using the formula stated in the methods13

a comparative graph of oxyzyme

hydrogel kill rates for all target species was constructed (fig 9.). This method takes

into account and corrects for any population changes that occur within the control

samples. All kill rates were significantly different from each other (p<0.0001), and

hence the order of sensitivity to the oxyzyme test dressings was; F. nucleatum, B.

fragilis, P. acnes, S. aureus (MRSA), S. epidermidis, C. albicans and Ps. aeruginosa.

S. epidermidis, Ps. aeruginosa, and C. albicans were treated with all three

hydrogel wound dressing formulations (oxyzyme, iodozyme 401 & iodozyme 402).

All wound dressings gave kill rates significantly different (p<0.0001) than either no

change (flat line) or either of the two control conditions, and all kill rates were

significantly different from each other (p<0.0001). The order of efficacy of the

hydrogel dressings was the same regardless of the target species; iodozyme

402>iodozyme 401>oxyzyme (table 2).

Discussion

12

The findings from this study point the way to further developments of the realistic, in-

vitro wound bed model, as well as helping to refine the mode of use and further

optimisation of the wound dressings themselves.

For example, it is helpful to understand the applicability of the low iodine

oxyzyme system (for use mainly as a prophylactic to minimise the risk of infection) in

contrast to the higher iodine iodozyme variants designed for use on infected wounds.

Although all of test dressings exerted a significant antimicrobial effect against all

target species, they did so with different potency (as expected). The oxyzyme

hydrogel dressing was most effective against the strict anaerobes B. fragilis and F.

nucleatum, followed by the aero tolerant anaerobe P. acnes. As might be expected

with anaerobes, aerobic conditions alone (control 1 and 2) produced a killing effect.

However, in all cases these rates were considerably less than that induced by the

active test dressing and hence the difference between control and test rates can be

ascribed to the antimicrobial efficacy of the dressing. It was apparent that control 1

(iodide plus glucose wound contact layer without the surface enzyme layer) produced

a higher kill rate than control 2, possibly due to the inactive iodide in the test dressing

being slowly oxidised by redox activity of the test microbial cells, yielding traces of

iodine. The marked efficacy of the oxyzyme hydrogel dressing against anaerobes is of

particular clinical significance, in the light of increasing clinical publications

correlating the prevalence of anaerobic bacteria with wound infection1.

Oxyzyme hydrogels were significantly effective against S. epidermidis and S.

aureus (MRSA), which is important in view of the recent emergence of antibiotic

resistant organisms, particularly S. aureus within wounds. This has heightened the

need to find effective prophylactic treatment that can reduce the colonisation and/or

the bacterial load of wounds by antibiotic resistant organisms.

13

Although fungal infections of skin wounds are much less common than those

associated with bacteria14

, the increase in antifungal resistance shown by C. albicans15

makes this an interesting target species to test against the oxyzyme hydrogels. The

killing of the eukaryotic C. albicans was less effective than that measured against the

prokaryotic anaerobes and staphylococci, although it was still significant. The more

resistant nature of C. albicans (in comparison with staphylococci) has also been

observed previously in photodynamic killing using methylene blue and light16

.

The lower efficacy of the oxyzyme hydrogel against Ps. aeruginosa, (with

regrowth after a killing phase of 6½ hours) is consistent with the known robustness of

this organism. Ps. aeruginosa has long been known to be a persistent and versatile

opportunistic pathogen that can adapt to and utilise numerous environmental

conditions – a capability that strengthens its pathogenic potential in wounds

(particularly burns), often associated with transmissible virulence factors17

. Although

the oxyzyme hydrogel system had limited antimicrobial efficacy against Ps.

aeruginosa, both of the iodozyme hydrogel systems (401 & 402) exerted significant

and prolonged antimicrobial activity, with no occurrence of re-growth. This suggests

that the increased generation of iodine within the hydrogel dressings increases the

antimicrobial power. This association between increased iodine generation capacity

and antimicrobial power was also evident in the experiments with iodozyme hydrogel

dressings tested against S. epidermidis12

and C. albicans. In all cases a significant

difference was seen between all three hydrogel dressings, with increasing

antimicrobial power relating to iodide content.

Conclusions

14

Many skin surface dressings now available incorporate antiseptics including iodine,

chlorhexidine or silver compounds. However the oxyzyme and iodozyme hydrogels

use antimicrobial systems that integrate active oxygenation with controlled synthesis

and release of iodine. The correction of wound-hypoxia by delivery of oxygen to the

wound promotes natural antimicrobial effects (especially via enhanced leukocyte

activity) and biochemical healing processes, all within a moist wound environment,

resulting in a plurality of antimicrobial effects.

In view of the demonstrated effective antimicrobial properties of these novel

hydrogel dressings, their use in the clinical environment could be significant in

reducing the microbial load of a wound, thereby limiting or inhibiting infection. They

are broad spectrum in activity, including antibiotic resistant organisms, anaerobes and

yeasts, and they were found to be rapidly effective. These dressings could

significantly enhance the treatment of wounds which are particularly prone to

complications resulting from infection. This offers potential benefits to the patient

through decreased trauma, and could significantly reduce treatment costs.

There are limitations with the in vitro model used in that the host response is

not taken into account, and so will not determine wound events or healing but merely

the propensity of the dressings to kill potentially pathogenic wound infecting

microbes. Hence, further testing is needed to determine the effectiveness of these

dressings at limiting the colonisation of potentially pathogenic organisms within the

wound environment in vivo. Further developments of the in-vitro model are to be

undertaken, especially as a means through which to optimise the performance of the

dressings.

Acknowledgements

15

The authors would like to thank Insense Ltd., Bedford, UK for supplying various

hydrogel test dressings and for summer studentship financial support (for RMS

Thorn).

References

16

1. Bowler PG, Duerden BI, Armstrong DG. Wound microbiology and associated

approaches to wound management. Clinical Microbiology Reviews 2001; 14: 244-69.

2. Revathi G, Puri J, Jain BK. Bacteriology of wounds. Burns 1998; 24: 347-9.

3. Ugburo AO, Atoyebi OA, Oyeneyin JO, Sowemimo GOA. An evaluation of the

role of systemic antibiotic prophylaxis in the control of burn wound infection at the

Lagos University Teaching Hospital. Burns 2004; 30: 43-8.

4. Wright JB, Lam K, Burrell RE. Wound management in an era of increasing

bacterial antibiotic resistance: A role for topical silver treatment. American Journal of

Infection Control 1998; 26: 572-7.

5. Hutchinson JJ, Lawrence JC. Wound infection under occlusive dressings. Journal

of Hospital Infection 1991; 17: 83-94.

6. Lawrence JC. Dressings and wound infection. The American journal of Surgery

1994; 167: S21-4.

7. Bowler PG, Jones SA, Davies BJ, Coyle E. Infection control properties of some

wound dressings. Journal of Wound Care 1999; 8: 499-502.

8. White RJ, Copper R, Kingsley A. Wound colonisation and infection: the role of

topical antimicrobials. British Journal of Nursing 2001; 10: 563-78.

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9. Drosou A, Falabella A, Kirsner RS. Antiseptics on wounds: An area of controversy.

Wounds 2003; 15: 149-66.

10. Wheeler JC, Woods JA, Cox MJ et al. Evolution of hydrogel polymers as contact

lenses, surface coatings, dressings, and drug delivery systems. Journal of Long Term

Effects of Medical Implants 1996; 6: 207-17.

11. Gates JL, Holloway GA. A comparison of wound environments. Ostomy Wound

Management 1992; 38: 34-7.

12. Thorn RMS, Greenman J, Austin A. In vitro method to assess the antimicrobial

activity and potential efficacy of novel types of wound dressings. Journal of Applied

Microbiology 2005; 99: 895-901.

13. Leeming, LP, KT Holland and RA Bojar. The in vitro antimicrobial effect of

azelaic acid. British Journal of Dermatology 1986; 115: 551-556

14. Mayhall CG. The epidemiology of burn wound infections: then and now. Clin

Infect. Dis. 2003; 37: 543-50.

15. Rex JH, Rinaldi MG, Pfaller MA. Resistance of Candida species to fluconazole.

Antimicrob Agents Chemother 1995; 39: 1-8.

16. Zeina B, Greenman J, Purcell WM, Das B. Killing of cutaneous microbial species

by photodynamic therapy. British Journal of Dermatology 2001; 144: 274-8.

18

17. Lyczak JB, Cannon CI, Pier GB. Establishment of Pesudomonas aeruginosa

infection: lessons from a versatile opportunist. Microbes and Infection 2000; 2:1051-

60.

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TABLES

Table 1. Kill rates and equivalent D-values for target species treated with the

oxyzyme hydrogel dressing.

Test Organism

Kill Rate (K = slope ± SD)

(log cfu reduction h-1

)

D Value (h)

S. epidermidis

S. aureus (MRSA)

Ps. aeruginosa

C. albicans

P. acnes

B. fragilis

F. nucleatum

-0.346 ± 0.040

-0.312 ± 0.084

-0.160 ± 0.030

-0.223 ± 0.010

-0.485 ± 0.034

-1.322 ± 0.297

-3.657 ± 0.299

2.887

3.201

6.262

4.466

2.061

0.756

0.273

20

Table 2. Kill rates for three target species treated with three different hydrogel

dressing formulations.

Test Organism

Test Dressing Kill Rate (K = slope)

(log cfu reduction h-1

)

D Value

(h)

S. epidermidis*

Ps. aeruginosa

C. albicans

Oxyzyme

Iodozyme 401

Iodozyme 402

Oxyzyme

Iodozyme 401

Iodozyme 402

Oxyzyme

Iodozyme 401

Iodozyme 402

-0.342 ± 0.054

-1.338 ± 0.245

-2.544 ± 0.330

-0.197 ± 0.035

-0.3873 ± 0.036

-0.695 ± 0.073

-0.223 ± 0.010

-0.996 ± 0.162

-1.299 ± 0.228

2.924

0.747

0.393

5.065

2.582

1.440

4.466

1.004

0.770

*Data from Thorn et al.12

21

Figure labels

Figure 1. Schematic diagram of Oxyzyme and Iodozyme dressings showing the

utilisation of glucose oxidase to drive the transport of oxygen into the wound, and the

synthesis in-place of iodine.

Figure 2. Cross-section schematic diagram of the experimental in vitro static diffusion

method. Test organisms are held within cellulose disks in close but indirect contact

with the dressing under test, and are exposed to any antimicrobial effects for defined

periods of time and under controlled physiochemical conditions. In addition, the

organisms held in the disk are also influenced positively by the water and solutes in

the nutrient agar, especially as they are drawn through the disks into the dressing by

fluid absorption. Quantifiable kill rates can then be accurately determined (previously

described by Thorn et al.12

).

Figure 3. Death kinetics of S. aureus (MRSA) treated with oxyzyme hydrogel. Test

treated with active oxyzyme hydrogel (♦), control treated with inactive hydrogel (□),

untreated control (∆), hatched line shows minimum level of detection. Line of best fit

on the test data relates to a linear regression of data points.

Figure 4. Death kinetics of Ps. aeruginosa treated with three hydrogel dressing

formulations. Test treated with oxyzyme hydrogel (♦), test treated with active

iodozyme 401 hydrogel (▼), test treated with active iodozyme 402 hydrogel (▲),

control treated with inactive hydrogel (□), untreated control (∆), hatched line shows

minimum level of detection. Lines of best fit on the test data relate to a linear

regression of data points.

22

Figure 5. Death kinetics of Candida albicans treated with three hydrogel dressing

formulations. Symbols as described in figure 2.

Figure 6. Death kinetics of P. acnes treated with a oxyzyme hydrogel. Symbols as

described in figure 1.

Figure 7. Death kinetics of B. fragilis treated with a oxyzyme hydrogel. Symbols as

described in figure 1.

Figure 8. Death kinetics of Fusobacteium nucleatum treated with a oxyzyme

hydrogel. Symbols as described in figure 1.

Figure 9. Comparative graph of the death kinetics of S. epidermidis (■), S. aureus

(MRSA) (♦), Ps. aeruginosa (●), C. albicans (�), P. acnes (�), B. fragilis (□), and

F. nucleatum (�) treated with a oxyzyme hydrogel dressing.

23

24

Figure 1.

Glucose oxidase

Glucose Gluconic Acid

Hydrogen Peroxide

2222

4444

Iodine

Iodide

Wound contact layer

Enzyme surface layer 1111

O2 O2

O2 O2 O2

1. Oxygen from the air is enzymically captured into the hydrogel by the action of glucose oxidase in the

enzyme surface layer. Oxygen is sparingly soluble in the hydrogel.

2. Glucose in the wound contact layer is oxidised to

gluconic acid with the production of hydrogen

peroxide which is used as a means of transporting

oxygen through the hydrogel layers .

3. Within the hydrogel matrix the hydrogen peroxide is consumed in a

complex reaction pathway with iodide ions resulting in oxygen being released

from the wound-facing surface.

3333

4. Iodine is also generated in this reaction pathway and is

gradually released from within the wound contact hydrogel

matrix.

O2 O2

O2 O2

O2

O2

Wound Surface

25

Figure 2.

26

Figure 3.

Cellulose disc carrying bacteria

Oxyzyme or Iodozyme test dressing

Comprising a glucose and iodide wound contact layer (yellow) and an enzyme

surface layer (red)

At set times, the discs are removed and the numbers of surviving

microbes determined

Quantification of surviving microbes by serial dilution and

spiral plating

Nutrient agar substrate

Control Samples

Comprising the glucose and iodide wound contact layer alone (yellow)

10-1

10-2

Nutrient agar substrate

10-1

10-2

27

0 1 2 3 4 5 6 7 8 9 103.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Time (h)

Log

cfu

per

cm

-2

Figure 4.

28

0 1 2 3 4 5 6 7 8 9 103.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

Time (h)

Log

cfu

per

cm

-2

29

0 1 2 3 4 5 6 7 8 9 103.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Time (h)

Log

cfu

per

cm

-2

Figure 5.

30

0 1 2 3 4 5 6 7 83.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Time (h)

Log c

fu p

er c

m-2

Figure 6.

31

0 1 2 3 4 5 6 7 83.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Time (h)

Log c

fu p

er c

m-2

Figure 7.

32

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.03.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

Time (h)

Log c

fu p

er c

m-2

Figure 8.

33

0 1 2 3 4 5 6 7 8 9 10-4

-3

-2

-1

0

1

Time (h)

Log

10 [

Nt/

N0] t

est

- L

og

10 [

Nt/

N0] c

on

tro

l

Figure 9.