Microscopy visualisation confirms multi-species biofilms are ubiquitous in diabetic foot ulcers

Abstract

Increasing evidence within the literature has identified the presence of biofilms in chronic wounds and proposed that they contribute to delayed wound healing. This research aimed to investigate the presence of biofilm in diabetic foot ulcers (DFUs) using microscopy and molecular approaches and define if these are predominantly mono- or multi-species. Secondary objectives were to correlate wound observations against microscopy results in ascertaining if clinical cues are useful in detecting wound biofilm. DFU tissue specimens were obtained from 65 subjects. Scanning electron microscopy (SEM) and peptide nucleic acid fluorescent in situ hybridisation (PNA-FISH) techniques with confocal laser scanning microscopy (CLSM) were used to visualise biofilm structures. Next-generation DNA sequencing was performed to explore the microbial diversity. Clinical cues that included the presence of slough, excessive exudate, a gel material on the wound bed that reforms quickly following debridement, poor granulation and pyocyanin were correlated to microscopy results. Of the 65 DFU specimens evaluated by microscopy, all were characterised as containing biofilm (100%, P < 0·001). The presence of both mono-species and multi-species biofilms within the same tissue sections were detected, even when DNA sequencing analysis of DFU specimens revealed diverse polymicrobial communities. No clinical correlations were identified to aid clinicians in identifying wound biofilm. Microscopy visualisation, when combined with molecular approaches, confirms biofilms are ubiquitous in DFUs and form either mono- or multi-species biofilms. Clinical cues to aid clinicians in detecting wound biofilm are not accurate for use in DFUs. A paradigm shift of managing DFUs needs to consider anti-biofilm strategies.

Keywords: Biofilms; Diabetic foot ulcers; Fluorescent in situ hybridisation; Microscopy; Scanning electron microscopy.

Figure 1

Scanning electron micrograph of DFU obtained from four patients demonstrating biofilm structure. (A) and (B) show large micro‐colonies of predominantly coccoid microbial cells encased in thick extracellular matrix (EPS) and anchored to collagen bundles within the wound (biofilm score 5). (C) shows large micro‐colonies of predominantly coccoid cells covered in a thin film of EPS (biofilm score 4). (D) Shows large micro‐colonies but less EPS (biofilm score 3).

Figure 2

CLSM of biofilm demonstrated via FISH and PNA‐FISH. (A) Patient 20, FISH with CLSM shows predominantly Gram‐negative rods in biofilm using green‐fluro‐488‐labelled probe targeting Pseudomonas spp. and yellow‐Cy3‐labelled universal bacterial probe. (B) Patient 4, PNA‐FISH with CLSM illustrates different bacterial morphologies of a multi‐species biofilm using fluorescent‐labelled universal PNA probes. (C) Patient 48, FISH with CLSM using red‐fluro‐488‐labelled universal probe is viewed at low magnification and illustrates the total amount of microbial biofilm on the tissue.

Figure 3

FISH and PNA‐FISH with CLSM technique to explore the spatial organisation of microbial aggregates in DFU samples. (A) Patient 18, identifies two mono‐species biofilm in the same wound, bacteria labelled with S. aureus PNA probe (green bacteria) and bacteria labelled with coagulase‐negative Staphylococci (red). (B) Patient 27, three‐dimensional view of a DFU biopsy depicted using the Imaris software. This highlights the structural complexity of biofilms where multi‐species biofilm coexist with mono‐species biofilms and planktonic microorganisms. (A) coagulase‐negative Staphylococci is red‐labelled PNA probe with (B) S. aureus, a green‐labelled PNA probe. (C) Mono‐species of S. aureus biofilm. (D) Planktonic aggregates of coagulase‐negative Staphylococci. (E) Planktonic aggregates of S. aureus. Top right corner viewing bubble demonstrates standard CLSM view of multi‐species biofilm under high magnification. (c). Patient 35, FISH with CLSM. Green arrow shows mono‐species biofilm (Staphylococcus spp. specific probe), and orange arrow shows mixed‐species biofilms (universal bacterial red probe).

Figure 4

Next‐generation DNA sequencing of 65 DFUs. Bar graphs depict the most common genera of microorganisms in DFUs. The vertical axis refers to relative abundance across DFUs. Horizontal axis is the participant number.