James Garth A, Ge Zhao Alice, Usui Marcia, Underwood Robert A, Nguyen Hung, Beyenal Haluk, deLancey Pulcini Elinor, Agostinho Hunt Alessandra, Bernstein Hans C, Fleckman Philip, Olerud John, Williamson Kerry S, Franklin Michael J, Stewart Philip S
Center for Biofilm Engineering, Montana State University, Bozeman, Montana.
Division of Dermatology, Department of Medicine, University of Washington, Seattle, Washington.
Wound Repair Regen. 2016 Mar;24(2):373-83. doi: 10.1111/wrr.12401. Epub 2016 Feb 16.
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.
生物膜与伤口愈合延迟有关，尽管生物膜损害伤口愈合的机制尚不清楚。许多细菌种类会产生可能抑制愈合的外毒素和外酶。此外，生物膜和反应性白细胞消耗氧气，可能会耗尽愈合所需的氧气，从而阻碍伤口愈合。在本研究中，使用了氧微传感器来测量通过体外培养的生物膜、糖尿病（db/db）小鼠伤口模型结痂内形成的体内生物膜以及离体人类慢性伤口标本的氧剖面。结果表明，小鼠结痂内的氧水平具有陡峭的梯度，在活体小鼠上最小值范围为17至72 mmHg，在安乐死小鼠上为6.4至1.1 mmHg。小鼠结痂中的氧梯度与体外培养的临床分离株和人类离体标本中观察到的相似。为了表征小鼠结痂中细菌的代谢活性，对与db/db小鼠伤口相关的铜绿假单胞菌生物膜进行了转录组学分析。结果表明，细菌表达了与细胞生长相关的代谢活性基因。有趣的是，转录组结果还表明伤口内的细菌经历了氧限制应激。在体内表达的细菌基因中，有与Anr介导的缺氧应激反应相关的基因。在体内高表达的其他细菌应激反应基因是与稳定期生长、渗透应激和RpoH介导的热休克应激相关的基因。总体而言，结果支持了这样的假设，即慢性伤口中的细菌生物膜通过其代谢活动以及通过募集为宿主防御过程消耗氧气的细胞，导致局部低氧张力的维持，从而促进慢性化。
