Nonrandom distribution of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds

Abstract

The spatial organization of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds was investigated in the present study. Wound biopsy specimens were obtained from patients diagnosed as having chronic venous leg ulcers, and bacterial aggregates in these wounds were detected and located by the use of peptide nucleic acid-based fluorescence in situ hybridization and confocal laser scanning microscopy (CLSM). We acquired CLSM images of multiple regions in multiple sections cut from five wounds containing P. aeruginosa and five wounds containing S. aureus and measured the distance of the bacterial aggregates to the wound surface. The distance of the P. aeruginosa aggregates to the wound surface was significantly greater than that of the S. aureus aggregates, suggesting that the distribution of the bacteria in the chronic wounds was nonrandom. The results are discussed in relation to our recent finding that swab culturing techniques may underestimate the presence of P. aeruginosa in chronic wounds and in relation to the hypothesis that P. aeruginosa bacteria located in the deeper regions of chronic wounds may play an important role in keeping the wounds arrested in a stage dominated by inflammatory processes.

FIG. 1.

Sampling region on a chronic venous leg ulcer. Biopsy specimens were taken from a central region within the wounds. The arrows point to a representative sampling region.

FIG. 2.

Distribution of the distances from the wound surface to the center of mass of S. aureus aggregates (light gray shading) or P. aeruginosa aggregates (dark gray shading). The distances are average values obtained from the analysis of 15 images for each wound sample.

FIG. 3.

Representative CLSM images of S. aureus (A and B), P. aeruginosa (C and D), and both organisms (E) in chronic wounds. The bacteria were detected by PNA-FISH with an FITC-labeled S. aureus-specific probe (green) or a TxR-labeled P. aeruginosa-specific probe (red), or a mixture of the two probes. Arrows point to the wound surfaces. Bars, 30 μm.

FIG. 4.

Epifluorescence micrographs (A and B) and bright-field micrographs (C and D) showing red PNA-FISH-stained P. aeruginosa cells (A), green PNA-FISH-stained S. aureus cells (B), blue DAPI-stained host cells (A and B), H&E-stained host cells and P. aeruginosa cells (C), and H&E-stained host cells and S. aureus cells (D). Some of the bacteria and host cells are encircled and labeled b and h, respectively. Arrows point to the wound surfaces. Bars, 35 μm.