Distribution, organization, and ecology of bacteria in chronic wounds

Abstract

Between 1 and 2% of the population in the developed world experiences a nonhealing or chronic wound characterized by an apparent arrest in a stage dominated by inflammatory processes. Lately, research groups have proposed that bacteria might be involved in and contribute to the lack of healing of these wounds. To investigate this, we collected and examined samples from chronic wounds obtained from 22 different patients, all selected because of suspicion of Pseudomonas aeruginosa colonization. These wound samples were investigated by standard culturing methods and peptide nucleic acid-based fluorescence in situ hybridization (PNA FISH) for direct identification of bacteria. By means of the culturing methods, Staphylococcus aureus was detected in the majority of the wounds, whereas P. aeruginosa was observed less frequently. In contrast, using PNA FISH, we found that a large fraction of the wounds contained P. aeruginosa. Furthermore, PNA FISH revealed the structural organization of bacteria in the samples. It appeared that P. aeruginosa aggregated as microcolonies imbedded in the matrix component alginate, which is a characteristic hallmark of the biofilm mode of growth. The present investigation suggests that bacteria present within these wounds tend to be aggregated in microcolonies imbedded in a self-produced matrix, characteristic of the biofilm mode of growth. Additionally, we must conclude that there exists no good correlation between bacteria detected by standard culturing methods and those detected by direct detection methods such as PNA FISH. This strongly supports the development of new diagnostic and treatment strategies for chronic wounds.

FIG. 1.

(a and b) Microcolonies of P. aeruginosa (identified by a specific PNA FISH probe [red stain]) surrounded by host cells (DAPI [blue stain]). (c) CLSM three-dimensional image of frame b. (d) Enlargement of frame c. (e) Large microcolony of S. aureus. (f) Single cells of S. aureus (identified by a specific PNA FISH probe [green stain]). Host cells are present (interspaced) (DAPI [blue stain]). (g) Microcolony of coccus-shaped bacteria (identified by a universal bacterial probe [green stain; appears purple here due to being mixed with the blue DAPI stain]). (h) Biofilm with mixed bacterial species (red stained bacteria are P. aeruginosa, and green stained bacteria are unidentified bacteria). White arrows point to bacterial aggregates (single cells in frame f), and yellow arrows point to the wound surface.

FIG. 2.

Presence of exopolymeric substance alginate within and surrounding P. aeruginosa aggregates, as detected by alginate-specific immunostaining. (A) Wound; (B) cystic fibrosis lung; (C) in vitro smear of mucoid P. aeruginosa; (D) in vitro smear of alginate-negative P. aeruginosa.

FIG. 3.

Number of wounds in each category. Among the wounds with evidence of P. aeruginosa colonization, P. aeruginosa was detected more frequently with PNA FISH (P < 0.02). This was in contrast to the higher frequency of S. aureus detected by culture growth (P < 0.0001) from the wounds with evidence of S. aureus. *, aggregates as described in the legend to Fig. 1.