Bacterial Aggregates Establish at the Edges of Acute Epidermal Wounds

Abstract

Objective: The bacterial composition and distribution were evaluated in acute standardized epidermal wounds and uninjured skin by a molecular in situ technology benchmarked to conventional culturing. This was done to reveal whether bacterial biofilm is present in acute wounds. Approach: On the buttock of 26 healthy volunteers, 28 suction blisters were made and de-roofed. Four wounds were biopsied immediately after wounding, whereas the remaining 24 wounds were treated daily with sterile deionized water and covered with a moisture-retaining dressing. On day 4 post-wounding, swabs were obtained for culturing from the wounds and adjacent skin, and the wounds including adjacent skin were excised. Tissue sections were stained with peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) probes, counterstained by 4',6-diamidino-2-phenylindole, and evaluated by confocal laser scanning microscopy (CLSM). Results: No bacterial aggregates were detected at day 0. At day 4, coagulase-negative staphylococci (CoNS) were the sole bacteria identified by CLSM/PNA-FISH and culturing. CoNS was isolated from 78% of the wound swabs and 48% of the skin swabs. Bacterial aggregates (5-150 μm) were detected by PNA-FISH/CLSM in the split stratum corneum and fibrin deposits at the wound edges and in the stratum corneum and the hair follicles of the adjacent skin. The bacterial aggregates were more common (p = 0.0084) and larger (p = 0.0083) at wound edges than in the adjacent skin. Innovation: Bacterial aggregates can establish in all wound types and may have clinical significance in acute wounds. Conclusion: Bacterial aggregates were observed at the edges of acute epidermal wounds, indicating initiated establishment of a biofilm.

Keywords: PNA-FISH; acute wounds; bacterial aggregates; biofilm; confocal microscopy; standardized epidermal wounds.

Thomas Bjarnsholt, DrMedSc, PhD

FIG. 1.

Bacterial aggregates establish at the wound edge. Skin sections stained by (a) hematoxylin and eosin showing the split stratum corneum and fibrin residue (black arrow) at the border of the wound bed (black bracket a) and the adjacent covered healthy skin (black bracket a). The same skin wound stained by fluorescent dyes (b) showing the location of the detail photo (white square) of (c) Bacterial aggregates (white arrows c) appear pink due to the red conjugated PNA probe and DAPI (blue). Nucleated eukaryotes are stained blue (black arrow c). Erythrocytes appear yellow (white arrow b) and surrounding tissues green due to auto-fluorescence. DAPI, 4′,6-diamidino-2-phenylindole; PNA, peptide nucleic acid.

FIG. 2.

Bacterial aggregates in adjacent skin covered by occlusive dressing. Bacterial aggregates indicated by arrows in a hair follicle (a) and on stratum corneum (b). Bacteria were stained by a universal bacterial-conjugated Texas Red (red) CoNS PNA probe and by DAPI (blue) and in combination they appear blue/purple due to low growth activity. Nucleated eukaryotes are stained blue, erythrocytes are yellow, and surrounding tissues appear green. CoNS, coagulase-negative staphylococci.

FIG. 3.

Distribution of the bacterial aggregates. (a) Distribution of aggregates according to size and location in wounds and adjacent skin post-wounding day 4 in 24 healthy volunteers. One symbol represents the largest aggregate diameter in each location per biopsy. In some biopsies, aggregates were observed in several locations. The dotted lines define the grouping of the aggregate sizes. (b) The aggregates diameter was measured in μm at the widest dimension.