Biofilms and Inflammation in Chronic Wounds

Abstract

Significance: The incidence, cost, morbidity, and mortality associated with non-healing of chronic skin wounds are dramatic. With the increasing numbers of people with obesity, chronic medical conditions, and an increasing life expectancy, the healthcare cost of non-healing ulcers has recently been estimated at $25 billion annually in the United States. The role played by bacterial biofilm in chronic wounds has been emphasized in recent years, particularly in the context of the prolongation of the inflammatory phase of repair.

Recent advances: Rapid high-throughput genomic approaches have revolutionized the ability to identify and quantify microbial organisms from wounds. Defining bacterial genomes and using genetic approaches to knock out specific bacterial functions, then studying bacterial survival on cutaneous wounds is a promising strategy for understanding which genes are essential for pathogenicity.

Critical issues: When an animal sustains a cutaneous wound, understanding mechanisms involved in adaptations by bacteria and adaptations by the host in the struggle for survival is central to development of interventions that favor the host.

Future directions: Characterization of microbiomes of clinically well characterized chronic human wounds is now under way. The use of in vivo models of biofilm-infected cutaneous wounds will permit the study of the mechanisms needed for biofilm formation, persistence, and potential synergistic interactions among bacteria. A more complete understanding of bacterial survival mechanisms and how microbes influence host repair mechanisms are likely to provide targets for chronic wound therapy.

Figures

Ge Zhao, MD, PhD

Figure 1.

Bacterial biofilm. Bacteria can attach to the tissue surface and form biofilm colonies, which have a different phenotype than planktonic bacteria. Tissue section of a diabetic mouse wound stained using Brown and Bren methods, which identifies both Gram-negative and Gram-positive bacteria shows formation of aggressive Pseudomonas aeruginosa biofilm. Scale bar=10 μm. ECM, extracellular matrix.

Figure 2.

Illustration shows presence of biofilm and leukocytes in a biofilm-challenged wound covered with a scab at 28 days post-wounding. (Adapted from Zhao et al.)

Figure 3.

Micrographs of P. aeruginosa on mouse wound. Light (a) and transmission electron microscopy (b) images show presence of biofilm containing rod-shaped P. aeruginosa (P) embedded in an extracellular matrix. (Adapted from Zhao et al.)

Figure 4.

Normal and delayed wound healing of cutaneous wounds. Illustration shows differences between normal healing of an excisional wound (a, b) compared to the delayed healing of an excisional wound in the presence of bacterial biofilm (c).

Figure 5.

Experimental overview of in vivo mouse wound model coupled with genome-wide mutant analysis (Tn-seq). (a) A pool of genome-wide transposon bacterial mutants is inoculated onto diabetic mouse wounds that are harvested at selected duration(s) of infection. Bacterial mutants lacking functions required for initiating infection are lost early during infection, and mutants unable to persist in wounds will be lost after a longer duration. Only bacterial mutants that did not lose an essential function were able to survive into the harvest pool of bacteria. (b) Genomic DNA isolated from the wound harvest is processed and examined using Tn-seq methodology to map the exact location of disrupted bacterial genes and quantify the relative abundance of individual mutants. The chromosome–transposon junctions are used as unique barcodes to identify each bacterial mutant. The relative abundance of individual bacterial mutants corresponding to specific Tn insertions is shown in the bar graph (height of blue and red columns). Whereas transposon insertion mutants in Gene B (orange shade) were abundant in the inoculum (blue columns), they were not detected in the harvest pool of bacteria. The encoded product of Gene B represents bacterial functions required for survival on the harvested wound. These bacterial functions inactivated by transposon insertion in their corresponding genes are potential novel therapeutic targets for chronic wound infections. Tn-seq, transposon-sequencing.