Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds.

Abstract

Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms and by the responding leukocytes, may impede wound healing by depleting the oxygen that is required for healing. In this study, oxygen microsensors to measure oxygen transects through in vitro cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse wound model, and ex vivo human chronic wound specimens was used. The results showed that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17 to 72 mmHg on live mice and from 6.4 to 1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. To characterize the metabolic activities of the bacteria in the mouse scabs, transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds was performed. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results also indicated that the bacteria within the wounds experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results supported the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions, through their metabolic activities and through their recruitment of cells that consume oxygen for host defensive processes.