Development of a High-Throughput ex-Vivo Burn Wound Model Using Porcine Skin, and Its Application to Evaluate New Approaches to Control Wound Infection.
Development of a High-Throughput ex-Vivo Burn Wound Model Using Porcine Skin, and Its Application to Evaluate New Approaches to Control Wound Infection.
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DOI：
10.3389/fcimb.2018.00196
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发表时间：
2018
影响因子：
5.7
通讯作者：
Jones BV
中科院分区：
文献类型：
--
作者：
Alves DR;Booth SP;Scavone P;Schellenberger P;Salvage J;Dedi C;Thet NT;Jenkins ATA;Waters R;Ng KW;Overall ADJ;Metcalfe AD;Nzakizwanayo J;Jones BV
伤口中的生物膜形成被认为是成功治疗的主要障碍，并且与伤口向慢性不愈合状态的转变有关。在这里，我们提出了一种新的实验室模型的伤口生物膜形成使用离体猪皮和定制的烧伤伤口阵列设备。该模型支持生物膜形成的高通量研究，并与一系列用于监测细菌生长，生物膜形成和基因表达的既定方法兼容。我们通过评估噬菌体控制金黄色葡萄球菌生物膜形成的潜力，以及群体密度依赖性的金黄色葡萄球菌表达的潜力，证明了该模型的应用。金黄色葡萄球菌毒力因子（由辅助基因调节因子agr调节）来发出临床相关伤口感染的信号。使用XTT测定法计数菌落形成单位和代谢活性，证实了伤口中细菌的生长，并显示噬菌体处理后活细胞显著减少。共聚焦激光扫描显微镜证实了伤口中生物膜的生长，并显示噬菌体治疗可以显着减少这些群落的形成。通过qRT-PCR对agr活性的评价显示，对于大多数菌株，在伤口模型中生长期间活性增加。设计用于提供伤口感染视觉信号的原型感染响应敷料的激活与agr活性增加有关。在所有测定中，使用该模型在重复测定之间观察到极好的再现性。
Biofilm formation in wounds is considered a major barrier to successful treatment, and has been associated with the transition of wounds to a chronic non-healing state. Here, we present a novel laboratory model of wound biofilm formation using ex-vivo porcine skin and a custom burn wound array device. The model supports high-throughput studies of biofilm formation and is compatible with a range of established methods for monitoring bacterial growth, biofilm formation, and gene expression. We demonstrate the use of this model by evaluating the potential for bacteriophage to control biofilm formation by Staphylococcus aureus, and for population density dependant expression of S. aureus virulence factors (regulated by the Accessory Gene Regulator, agr) to signal clinically relevant wound infection. Enumeration of colony forming units and metabolic activity using the XTT assay, confirmed growth of bacteria in wounds and showed a significant reduction in viable cells after phage treatment. Confocal laser scanning microscopy confirmed the growth of biofilms in wounds, and showed phage treatment could significantly reduce the formation of these communities. Evaluation of agr activity by qRT-PCR showed an increase in activity during growth in wound models for most strains. Activation of a prototype infection-responsive dressing designed to provide a visual signal of wound infection, was related to increased agr activity. In all assays, excellent reproducibility was observed between replicates using this model.