Examples: histone, BN000065

Project: PRJNA276294

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, as well as responding leukocytes, may impede wound healing. In this study, we used oxygen microsensors to measure oxygen transects through in vitro-cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse model, and ex vivo human chronic wound specimens. The results show that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17-72 mmHg on live mice and 6.4-1.1 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. No oxygen gradients were observed for heat-killed mouse scabs, suggesting that active metabolism by the viable bacteria and host cells contributed to the reduced oxygen partial pressure of the scabs. To characterize the metabolic activities of the bacteria in the mouse scabs, we performed transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds using Affymetrix microarrays. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results indicated that the bacteria within the wounds also 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 support the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions. Overall design: The following procedure was performed on two mice, resulting in two biological replicates. A punch biopsy wound was created on the dorsal surface of a diabetic mouse, subsequently infected with a P. aeruginosa biofilm two days post-wounding, and covered with semi-occlusive dressings for two weeks. At 28 days post wounding, the wound crust was surgically excised and RNA was extracted for analysis on Affymetrix P. aeruginosa microarrays. To conservatively identify the most abundant transcripts in the mouse wound scabs, the transcripts with the highest signal intensities (the top 5% of the transcripts represented on the arrays) were selected for further analysis. We sought to characterize the persistence of P. aeruginosa activity within biofilms in the mouse wound model by isolating and identifying mRNA from the wounds 28 days post infection.

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