An in vitro test of the efficacy of an anti-biofilm wound dressing

https://doi.org/10.1016/j.ijpharm.2014.08.034Get rights and content

Abstract

Broad-spectrum antimicrobial agents, such as silver, are increasingly being formulated into medicated wound dressings in order to control colonization of wounds by opportunistic pathogens. Medicated wound dressings have been shown in-vitro to be effective against planktonic cultures, but in-vivo bacteria are likely to be present in biofilms, which makes their control and eradication more challenging. Recently, a functional wound dressing (AQUACEL® Ag+ Extra™ (AAg + E)) has been developed that in addition to silver contains two agents (ethylenediaminetetraacetic acid (EDTA) and benzethonium chloride (BC)) designed to disrupt biofilms. Here, the efficacy of AAg + E is demonstrated using a biofilm model developed in an isothermal microcalorimeter. The biofilm was seen to remain viable in the presence of unmedicated dressing, silver-containing dressing or silver nitrate solution. In the presence of AAg + E, however, the biofilm was eradicated. Control experiments showed that neither EDTA nor BC alone had a bactericidal effect, which means it is the synergistic action of EDTA and BC disrupting the biofilm with silver being bactericidal that leads to the product’s efficacy.

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.

References (31)

  • E. Banin et al.

    Chelator-induced dispersal and killing of Pseudomonas aeruginosa cells in a biofilm

    Appl. Environ. Microbiol.

    (2006)
  • A.E. Beezer et al.

    Application of flow microcalorimetry to analytical problems–preparation, storage and assay of frozen inocula of saccharomyces cerevisiae

    J. Appl. Bact.

    (1976)
  • P.G. Bowler

    Wound pathophysiology: infection and therapeutic options

    Ann. Med.

    (2002)
  • P.G. Bowler et al.

    Wound microbiology and associated approaches to wound management

    Clin. Microbiol. Rev.

    (2001)
  • P.G. Bowler et al.

    Microbicidal properties of a silver-containing Hydrofiber® dressing against a variety of burn wound pathogens

    J. Burn Care Rehabil.

    (2005)
  • Cited by (28)

    • Engineering an integrated electroactive dressing to accelerate wound healing and monitor noninvasively progress of healing

      2022, Nano Energy
      Citation Excerpt :

      Supplementary material related to this article can be found online at. Infections represent a critical challenge for wound care; they typically involve the presence of aggregated bacteria that are embedded in a self-secreted extracellular matrix, forming a biofilm [46,47]. Biofilms on wounds generate chronic infections, so preventing the formation of a biofilm is an important aspect of effective wound care [46,47].

    • The efficacy of topical agents used in wounds for managing chronic biofilm infections: A systematic review

      2020, Journal of Infection
      Citation Excerpt :

      In seventeen studies, biofilm growth pre-testing was not visualised by any microscopy method, with biofilm growth assumed (43%, 17 of 39).29,36,37,39,40,47,50,52,54,58,59,61 –66 A control or untreated sample was used in thirty-two studies, with seven studies having no study control.28,32,34,37,44,52,63 The most frequent control was an untreated sample, (51%, 20 of 39) while a positive and negative control was used in seven studies (18%, 7 of 39).54,64–69

    • Use of a water-based probiotic to treat common gut pathogens

      2019, International Journal of Pharmaceutics
      Citation Excerpt :

      In this work we report the evaluation of the antipathogenic activity of Symprove against three common infectious pathogens; Escherichia coli, Shigella sonnei and MRSA. Isothermal calorimetry, a technique that has been used previously in the study of bacterial growth kinetics was used here for analysis to provide real-time data on microbial growth kinetics (Said et al., 2014a; Said et al., 2014b; Braissant et al., 2010; Fredua-Agyeman and Gaisford, 2015; Fredua-Agyeman et al., 2017). Symprove (original flavour) was supplied by Symprove Ltd (Farnham, UK).

    • Development of a flow system for studying biofilm formation on medical devices with microcalorimetry

      2015, Methods
      Citation Excerpt :

      For instance, Clauss et al. [30] monitored biofilms of Staphylococcus aureus growing on human and bovine bone grafts, while Astasov-Frauenhoffer et al. [31] investigated variability and dynamics of a triple-species biofilm and determined efficacy of amoxicillin and metronidazole combinations against biofilms of various species [32]. Said et al. [33] determined the efficacy and mechanism of action of an anti-biofilm wound dressing. Lerchner et al. [34] note, however, that despite the widespread use of IMC for studying planktonic cells, its use for biofilm investigation is rarely considered.

    View all citing articles on Scopus
    View full text