by Cecilie Christiansen and  Marie Allesen-Holm

Introduction

Clinically, implementation of biofilm based wound management has recently gained increasing attention⁴ and ideally, evaluation of antimicrobial wound dressings should include biofilm models aswell as standard antimicrobial tests. Biatain Silicone Ag and Biatain Ag were tested in two different in vitro test methods, a wound biofilm model and a standard antimicrobial test over time. As biofilms in non-healing wounds are heterogeneously distributed in the wound, including in the tissue below the wound bed ³, Biatain Silicone Ag and Biatain Agwere evaluated in an in vitro wound biofilm model that specifically addresses the problematic biofilms heterogeneously embedded in the wound environment. The study was published at EWMA 2018²⁰

Wound biofilm model

The aim of this test was to evaluate the efficacy of Biatain Silicone Ag and Biatain Ag against maturebiofilms and in the prevention of biofilm formation in a biofilm model simulating biofilms embedded inthe wound environment.

Methods

The in vitro wound biofilm model (WBM) is based on a study by S. Crone et al. and was developed at Costerton Biofilm Center, University of Copenhagen ³⁰. The model consists of biofilm aggregates (either P. aeruginosa or S. aureus) embedded and grown in semi-solid agar. S. aureus and P. aeruginosa are both keen biofilm formers and will form mature biofilms within 24 hours in vitro. The microorganisms were inoculated into the semi-solid agar containing nutrients and either grown to mature biofilms for 24 hours or treated shortly after inoculation to demonstrate biofilm prevention. In both test setups, the microorganisms/biofilms were subsequently exposed for 24 hours to samples of Biatain Silicone Ag,Biatain Ag or control dressings without silver (Figure 1).

Model drawing of the WBM illustrating biofilms embedded in the agar. 

Figure 1

Results

Both Biatain® Silicone Ag and Biatain Ag showed statistically significant effect against mature biofilmsof S. aureus and P. aeruginosa, compared to control dressings without silver (Figure 2A & B). Both testdressings reduced mature P. aeruginosa biofilms by more than 99.99% and mature S. aureus biofilmsby 99.3% (Biatain Silicone Ag) and 99.93% (Biatain Ag), (p<0.001 vs. control for all, Students T-test).The variation in results between different bacterial strains is expected and caused by the differences insusceptibility of microorganisms to silver.

Killing of mature biofilms tested in the WBM. 

The results are shown as geometrical mean of CFU/ml ± standard deviation (SD). N=20 samples. The horizontal line represents limit of detection at 25 CFU/ml (CFU=Colony Forming Unit).

Figure 2

Biatain Silicone Ag and Biatain Ag equally prevented growth of biofilms of S. aureus and P. aeruginosa (p<0.001 vs. control for all, Students T-test) to the limit of detection which was set to 25 CFU/m l (Figure 3A & B).

Prevention of biofilm formation tested in the WBM. 

The results are shown as geometricalmean of CFU/ml ± SD. N=20 samples. The horizontal line represents limit of detection at 25 CFU/ml.

Figure 3

Discussion

Biatain® Silicone Ag and Biatain Ag were effective against mature biofilms and in prevention of biofilm formation. Both treatment of mature biofilms and prevention of biofilm formation are essential strategies in the framework for the treatment of wounds with biofilms ⁴. The differences in the efficacy against mature S. aureus and P. aeruginosa biofilms were expected and most likely caused by differences in susceptibility of the two microorganisms to silver. A generally accepted explanation to this, is the structural differences in the cell walls of Gram-positive and Gram-negative bacteria. Gram positive bacteria such as S. aureus have thicker cell walls that are more difficult for silver ions to penetrate^31,32. Additionally, microbiological variation also cause some variation in test results, e.g. as the difference seen for S. aureus and the two tested products.

Standard antimicrobial testing over 7 days

The “Standard test method for determining the antimicrobial activity of antimicrobial agents under dynamic contact conditions”, ASTM E2149-13a³¹ enables a simple, standard evaluation of antimicrobial wound dressings against a wide range of pathogenic microorganisms normally found in non-healing wounds at time points representing relevant wear times.

Method description

Tests of Biatain Silicone Ag and Biatain Ag were performed over a 7-day period. Dressing samples were submerged in separate Erlenmeyer flasks containing a microbial monoculture with a starting concentration of 105-106 CFU/ml. The samples were incubated for 24 hours and then moved to newflasks, every day for 7 days. This challenges the samples as it is repeatedly exposed to excessive liquid containing high concentration of microorganisms (Figure 4).

Illustration of the E2149-13a test methods. 

Figure 4

The antimicrobial activity was evaluated based on the log reduction results. The current log reduction requirements for antimicrobial wound dressings is defined as a log 3 reduction compared with the start concentration of microorganisms (prEN16756)³⁴

Six microorganisms were tested in the model, representing some of the most prevalent and pathogenic microorganisms found in infected wounds^35,37, including antibiotic resistant bacteria, and broadly covering the microbial differences between Gram-positive bacteria, Gram-negative bacteria and fungi:

• Staphylococcus aureus (Gram-positive bacteria)

• Pseudomonas aeruginosa (Gram-negative bacteria)

• Methicillin-resistant Staphylococcus aureus (MRSA) (Gram-positive bacteria)

• Vancomycin-resistant Enterococci (VRE) (Gram-positive bacteria)

• Candida albicans (yeast)

• Aspergillus brasiliensis (mold)

Results

Both dressings reduced all six tested microorganisms, including the antibiotic resistant strains, by morethan log 3. The antimicrobial activity was similar on day 1 and 7 (Figure 5 A & B) indicating asustained and effective release of silver up to 7 days. The variation in results between differentmicroorganisms is expected and caused by the differences in susceptibility of microorganisms to silverand general microbiological variation.

Antimicrobial efficacy tested according to ASTM E2149-13a against a broad range of microorganisms. 

Figure 5

Discussion

The ASTM E2149-13a test provides information about antimicrobial efficacy of a wound dressingagainst a broad range of planktonic microorganisms over time. The antimicrobial efficacy of both Biatain® Silicone Ag and Biatain Ag was sustained for 7 days with the daily challenge of new freshlycultured microorganisms.

The test represents a worst-case scenario. In non-healing wounds, new microorganisms would not besupplied every day and the amount of released silver would accumulate in the wound bed, resulting ina higher concentration of silver over time and a potentially greater antimicrobial effect than in the invitro situation. On the other hand, this standard test does not consider the presence and complexity ofmicrobial biofilms and only provides information about the basic antimicrobial activity. Therefore, thetest results should not be the sole basis for an antimicrobial product evaluation.

Conclusion on in vitro tests

Biatain Silicone Ag and Biatain Ag dressings are modern wound dressings containing the antimicrobial agent silver with an intended use for infected wounds and wounds at risk of infection. Biatain Silicone Agand Biatain Ag demonstrated antimicrobial efficacy against a wide range of pathogenic microorganisms commonly found in non-healing and infected wounds. The antimicrobial effect was continuous for 7 days with daily challenge of freshly cultured microorganisms. Biatain Silicone Ag and Biatain Ag also demonstrated statistically significant efficacy against mature biofilms of both S. aureus and P. aeruginosa, and in prevention of biofilm formation in an embedded wound biofilm model. Both treatment of mature biofilms and prevention of biofilm formation are essential strategies in the framework for the treatment of wound infection ⁴