An in vitro simulated chronic wound biofilm model used to assess the performance of silver-containing dressings against biofilm formation
Ruth Scully, Section Leader/Senior BMS, Electron Microscopy Unit, Cellular Pathology, Cardiff & Vale UHB, University Hospital of Wales; Dr Jan Hobot, Electron Microscopy Facility, Institute for Translation, Innovation, Methodology & Engagement (TIME), School of Medicine, Cardiff University; Mike Walker PhD, Independent Wound and Skin Care Consultant, Holywell, UK; Daniel Metcalf PhD, Associate Director, R&D, ConvaTec Ltd, Flintshire, UK.
The deleterious effect of microbes in wounds has been recognised for many years, and was originally thought to be simply due to the excessive presence of planktonic microorganisms. However, it is now accepted that microorganisms prefer to live in communal colonies referred to collectively as biofilm. In order to help reduce bioburden and the risk of infection, there are many antimicrobial wound dressings available, several of which contain silver. In this study, using a recently developed in vitro simulated chronic wound biofilm model, several of these silver-containing dressings have been investigated to evaluate their anti-biofilm potential.
A PET track-etched membrane cell culture insert in tissue culture wells was used as the biofilm substrate, as this has been previously shown to produce biofilm growth within 6 hours, which subsequently becomes established within a 24-48 hour period, as demonstrated by a variety of microscopic methods including light (LM), scanning electron (SEM) and transmission electron microscopy (TEM) (Scully et al, 2014).
Each insert contained a thin slice cut from a piece of sterile pork belly, and a 100 µl volume of a mixed bacterial culture (a 50 µl volume of a 3.4 x 107 cfu/ml Pseudomonas aeruginosa NCIMB 8626 culture and a 50 µl volume of a 3.4 x 107 cfu/ml Staphylococcus aureus NCIMB 9518 culture). Following application of the mixed culture, individual silver dressings (four different silver-containing dressings*) were then applied to evaluate whether biofilm could be prevented from growing beneath each dressing. Multiple plates were incubated with agitation at 37°C for 3, 6, 12, 24, 48 and 72 hours. At 24 hour intervals the external wells were flushed with a 1.5 ml volume of Tryptone Soya Broth to simulate wound exudate, and tissue and dressings were removed at the above time points for observations using LM (with haematoxylin & eosin (H&E) staining), SEM and TEM.
Image J open access image analysis software was used to carefully trace, with a computer mouse on screen, around those areas where stained biofilm could be observed after dressing removal. Total biofilm area (µm2) was then calculated from 10 random, representative LM images for each silver test dressing. Only central areas from each biofilm model that were exposed to the silver test dressings were selected.
Two-sample t-tests and Analyses of Variance (ANOVA) statistical tests were conducted using Minitab 17 software to compare the means of biofilm areas remaining beneath each silver test dressing.
Measureable biofilm was observed beneath some of the silver-containing dressings (blue areas in Figs 1-3), with the exception of the NGAD (Fig 4). Biofilm, where observed, was apparent in pockets – on the surface, suggesting limited direct contact with silver, and sometimes deeper within the tissue (Fig 1-3).
Some of these differences in silver dressing performance in this biofilm model, in terms of analysis of biofilm area reduction as an indicator of anti-biofilm activity, were statistically significant as demonstrated by t-tests (p<0.05) (Table 1). ACN-SD resulted in significantly less biofilm area on the samples than the NC-SD and CES-SD after 24 hours, and significantly less than CES-SD after 48 hours. The NGAD resulted in significantly less biofilm area on the samples than all other silver test dressings at all time-points (as exemplified by ANOVA at 72-hours in Fig 5).
Similar results were shown under SEM examination, with biofilm readily observed on the meat following removal of standard silver dressings (Fig 6A-C), and in one case, dressing fibres (Fig 6B), but only sporadic bacterial colonies following NGAD removal (Fig 6D), as exemplified by these 48-hour images.
Discussion & Conclusion
Some of the silver-containing dressings were shown to be statistically more effective than others in managing biofilm in this in vitro simulated chronic wound model. In particular dressing structure was demonstrated to be an influential factor in that two of the dressings (NC-SD and ACN-SD) had relatively open structures that allowed pockets of bacterial growth to take place on the meat surface, possibly due to limited intimate contact. These results are similar to a previous study that used agar plates to highlight the importance of dressings providing good intimate contact (Bowler et al, 2010). A third dressing (CES-SD) shed fibres upon removal, as well as allowing biofilm growth on the meat surface. Following removal of the fourth dressing (NGAD), which contains components designed to disrupt and prevent biofilm, minimal biofilm was observed beneath the dressing. This study provides further support to the clinical findings recently published (Walker et al, 2015; Metcalf et al, 2016) for this new dressing that is designed to disrupt biofilm, kill biofilm bacteria and prevent biofilm re-formation.
1. Scully et al. In vitro observations of biofilm formation beneath a range of antimicrobial wound dressings. Poster presented at Wounds UK 2014, Harrogate.
3. Bowler et al. Dressing conformability and silver-containing wound dressings. Wounds UK 2010; 6: 14-20.
4. Walker et al. A real-life clinical evaluation of a next-generation antimicrobial dressing on acute and chronic wounds. J Wound Care. 2015; 24: 11-22.
5. Metcalf DG, Parsons D, Bowler PG. A next-generation antimicrobial wound dressing: a real-life clinical evaluation in the UK and Ireland. J Wound Care 2016; 25: 132-138.