Clinical investigation of biofilm in non-healing wounds by high resolution microscopy techniques

Abstract

Objective: The aim of this study was to analyse wound biofilm from a clinical perspective. Research has shown that biofilm is the preferred microbial phenotype in health and disease and is present in a majority of chronic wounds. Biofilm has been linked to chronic wound inflammation, impairment in granulation tissue and epithelial migration, yet there lacks the ability to confirm the clinical presence of biofilm. This study links the clinical setting with microscopic laboratory confirmation of the presence of biofilm in carefully selected wound debridement samples.

Method: Human wound debridement samples were collected from adult patients with chronic non-healing wounds who presented at the wound care centre. Sample choice was guided by an algorithm that was developed based on what is known about the characteristics of wound biofilm. The samples were then evaluated by light microscopy and scanning electron microscopy for the presence of biofilm. Details about subject history and treatment were recorded. Adherence to biofilm-based wound care (BBWC) strategies was inconsistent. Other standard antimicrobial dressings were used and no modern antiseptic wound dressings with the addition of proven antibiofilm agents were available for use.

Results: Of the patients recruited, 75% of the macroscopic samples contained biofilm despite the prior use of modern antiseptic wound dressings and in some cases, systemic antibiotics. Wounds found to contain biofilm were not all acutely infected but biofilm was present when infection was noted. The clinical histories associated with positive samples were consistent with ideas presented in the algorithm used to guide sample selection.

Conclusion: Visual cues can be used by the clinician to guide suspicion of the presence of wound biofilm. This suspicion can be further enhanced with the use of a clinical algorithm. Standard antiseptic wound dressings used in this study demonstrated limited antibiofilm efficacy. This study also highlighted a need for the clinical team to focus on expiration of dressing action and consistent practice of BBWC strategies which includes the use of proven antibiofilm agents.

Keywords: biofilm; clinical; non-healing; wound.

Fig 1

Significantly ischemic left lateral leg traumatic ulcer. The chronic non-healing wound was treated with a polyurethane foam dressing containing silver nanoparticles that are purported to have antimicrobial activity. After removal of polyurethane foam dressing observed in (a), green film was present on the bed of this significantly ischaemic wound (b)

Fig 2

Analyses of chronic non-healing wound samples by microscopical techniques. Chronic non-healing wounds (macroscopic view) were subjected to sharp debridement. The tissue removed from debridement was fixed, sectioned and analyzed by either hematoxylin and eosin stain and light microscopy (H&E micrographs), or by high resolution field-emission gun scanning electron microscopy (high resolution scanning electron micrographs). H&E micrographs were collected at 100X and inset panels were collected at 400X total magnification. Arrows indicate region magnified in inset panel. High resolution scanning electron micrographs were collected between 2,000X and 20,000X

Fig 2

Analyses of chronic non-healing wound samples by microscopical techniques. Chronic non-healing wounds (macroscopic view) were subjected to sharp debridement. The tissue removed from debridement was fixed, sectioned and analyzed by either hematoxylin and eosin stain and light microscopy (H&E micrographs), or by high resolution field-emission gun scanning electron microscopy (high resolution scanning electron micrographs). H&E micrographs were collected at 100X and inset panels were collected at 400X total magnification. Arrows indicate region magnified in inset panel. High resolution scanning electron micrographs were collected between 2,000X and 20,000X

Fig 3

Analyses of chronic non-healing wound samples by microscopical techniques. Chronic non-healing wounds (macroscopic view) were subjected to sharp debridement. The tissue removed from debridement was fixed, sectioned and analysed by either hematoxylin and eosin stain and light microscopy (H&E micrographs), or by high resolution field-emission gun scanning electron microscopy (high resolution scanning electron micrographs). Samples 5, 7, 9,16 are taken from one patient over a number of weeks and following different treatments. H&E micrographs were collected at 100X and inset panels were collected at 400X total magnification. Arrows indicate region magnified in inset panel. High resolution scanning electron micrographs were collected between 2,000X and 20,000X

Fig 4

Analyses of chronic non-healing wound samples by microscopical techniques. Samples 6 and 12 are taken from one patient over a number of weeks and following different treatments. Chronic non-healing wounds (macroscopic view) were subjected to sharp debridement. The tissue removed from debridement was fixed, sectioned and analysed by either hematoxylin and eosin stain and light microscopy (H&E micrographs), or by high resolution field-emission gun scanning electron microscopy (high resolution scanning electron micrographs). H&E micrographs were collected at 100X and inset panels were collected at 400X total magnification. Arrows indicate region magnified in inset panel. High resolution scanning electron micrographs were collected between 2,000X and 20,000X

Fig 5

Analyses of chronic non-healing wound samples by microscopical techniques. Samples 8 and 15 are taken from one patient over a number of weeks and following different treatments. Chronic non-healing wounds (macroscopic view) were subjected to sharp debridement. The tissue removed from debridement was fixed, sectioned and analysed by either hematoxylin and eosin stain and light microscopy (H&E micrographs), or by high resolution field-emission gun scanning electron microscopy (high resolution scanning electron micrographs). H&E micrographs were collected at 100X and inset panels were collected at 400X total magnification. Arrows indicate region magnified in inset panel. High resolution scanning electron micrographs were collected between 2,000X and 20,000X

Fig 6

Evaluation of in vitro biofilm formation. High resolution scanning electron micrographs were collected between 2,000X and 10,000X total magnification (inset panel 20,000X) for comparison of in vitro biofilm with in vivo biofilm found within wound samples. Magnification bars indicate scale for each micrograph. White arrows indicate channels formed between clusters of cells that are likely used for diffusion of nutrients within the biofilm and dispersal of metabolic waste products out