An in vitro test of the efficacy of an anti-biofilm wound dressing
Graphical abstract
Introduction
The process of healing of chronic cutaneous wounds is complex and may be affected by the presence of microorganisms (Bowler, 2002). In particular, bacterial contamination may slow wound healing, resulting in damage to surrounding tissue and ultimately infection of the host (Landis, 2008). Progression to infection may also be aided by other factors including poor blood supply to the wound and the intrinsic virulence properties of the invading organisms (Siddiqui and Bernstein, 2010, Bowler et al., 2001).
The fact that the treatment of bacterial infection at the wound site has the potential significantly to reduce the time for wound healing has led to the development of a number of medicated wound dressings containing antimicrobial agents. Silver is a particularly widely used agent, as it shows broad antimicrobial (against both Gram-negative and Gram-positive organisms, Miraftab et al., 2014) and anti-fungal activity (Bowler et al., 2005), although there is debate as to the specific efficacy of silver (Aziz et al., 2012, White and Cutting, 2006) and to its potential toxicity (Hermans, 2006).
An added complexity in the treatment of chronic wound infections is that the organisms are frequently present as biofilms, with this form of bacterial growth being increasingly implicated in cases of poor wound healing (Hurlow and Bowler, 2012). Evidence for biofilm involvement in chronic wounds comes from macroscopic (Hurlow and Bowler, 2012, Metcalf and Bowler, 2013) and microscopic observation of biofilms (James et al., 2008, Metcalf and Bowler, 2013). A study involving in vivo wound models of a Staphylococcus aureus infection also showed microscopic evidence of biofilm formation along with a demonstration of physiological differences between planktonic cells and biofilm bacteria recovered from wounds (Davis et al., 2008).
While the drivers for biofilm formation are not completely clear, experimentally it is usually observed that microbial biofilms show reduced antimicrobial sensitivity than comparable plankonic organisms (Davis et al., 2008, Percival et al., 2011). Antibiotic resistance has been ascribed to several mechanisms, including the production of inactivating enzymes, the presence of persister cells and the protective effects of extracellular polymeric substances (EPS) (Smith, 2005).
Successful treatment of chronic wound infections therefore requires development of next generation of medicated dressings that are efficacious against biofilms. AQUACEL® Ag+ Extra™ (AAg+E) is a recently developed dressing that contains two agents that are known to disrupt biofilms, ethylenediaminetetraacetic acid (EDTA) and benzethonium chloride (BC), in addition to ionic silver as an antimicrobial agent. A recent cohort analysis of wound healing data involving 121 clinical cases showed AAg+E to result in progress toward healing in over 90% of wounds which were previously stalled, infected or at a risk of infection and with a high suspicion of biofilm contamination (Metcalf et al., 2014).
Determination of wound dressing efficacy using analytical or traditional microbiological methods is tricky, because of the challenges inherent in determining viable bacterial counts in a heterogeneous system. Isothermal microcalorimetry (IMC) is one technique that offers potential in this area, since it can detect the power resulting from bacterial growth without requiring optical clarity of the sample. We showed previously how IMC can be used to quantitate wound dressing efficacy (Gaisford et al., 2009, Said et al., 2014) against planktonic cultures of two common wound pathogens. To use IMC to investigate the efficacy of AAg+E against biofilms, however, requires development of a biofilm model. Hence, the specific aim of this work was to develop a biofilm model suitable for use with IMC and to use the model to explore the efficacy of AAg+E.
Section snippets
Material and methods
Ethylenediaminetetraacetic acid (EDTA), benzethonium chloride (BC) and silver nitrate (AgNO3) were purchased from Sigma (UK) and used as received. Wound dressings, AQUACEL® (AH), AQUACEL® Ag (AAgH) or AQUACEL® Ag Extra (AAg+E) were supplied by ConvaTec Ltd. The wound dressings, all comprised of sodium carboxymethylcellulose fibers, differ in that AH has no antimicrobial agent while AAgH contains ionic silver and AAg+E contains ionic silver, EDTA and BC.
The challenge organism, S. aureus NCIMB
Results and discussion
Although not discussed here, it was not possible to culture a biofilm directly onto the glass walls of the ampoule. Rather, the agar layer was necessary to encourage biofilm attachment and growth. A similar effect has been noted in ecological research where a twofold increase in the accumulation of diatoms (phytoplanktonic algae) was seen on a surface when unenriched agar was added (Stevenson, 1983). The rationale in that study was the use of agar to ‘simulate mucilage of immigrating
Summary
A biofilm model was developed that enabled the use of IMC to test the efficacy of an anti-biofilm wound dressing. It was shown that a broad spectrum antimicrobial agent alone was not effective against the biofilm but that when biofilm disrupting agents were included in the dressing bactericidal action was seen.
Funding
This work was supported by an Engineering and Physical Sciences Research Council CASE Award (grant number EP/H501398/1), partly funded by ConvaTec Ltd.
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