Biofilms and Bacteriophages in Chronic Wound Infections

项目摘要 铜绿假单胞菌是一种主要的人类病原菌，其毒力取决于其形成的能力。 生物膜--黏糊糊的多糖层和细菌层，使PA能够在伤口上定居，并逃避抗生素。 我的实验室最近发现了噬菌体在PA生物膜的形成和功能中扮演的新角色。我们 据报道，由PA产生的PF噬菌体(PF噬菌体)自发地组装宿主的松散网络 和存在于PA感染部位的微生物聚合物形成致密的、高度稳定的液晶。这颗水晶 结构提高了生物膜的粘附性、粘滞性和抗干性。 我们最近还报道，PF噬菌体和这种晶体结构通过以下方式促进抗生素耐受性 结合和隔离抗生素。这种效果取决于抗生素的电荷性质；肯定的 带电的抗生素，如妥布霉素，可以有效地隔离在带负电的结晶束中 噬菌体，而中性电荷的抗生素，如环丙沙星，则不是。这些数据表明Pf的存在 噬菌体可能是决定哪些抗生素对PA有效的重要因素。 总而言之，这些数据表明，PA细菌和PF噬菌体可能以某种方式配对，从而有助于 生物被膜感染的致病性。与此一致，我们的初步数据表明，在帕金森病的小鼠模型中 生物被膜伤口感染、PF噬菌体的存在导致慢性感染。 这些对PF噬菌体致病作用的新见解有可能开辟新的战线 对抗PA生物膜的斗争。然而，首先重要是确定它们与人类慢性伤口的相关性 感染。在目标1中，我们将评估人体伤口中的PA生物膜是否具有晶体结构并定义 Pf噬菌体水平与临床结果的关系，包括抗生素耐药性。 这些对生物被膜致病的贡献，可能有利于靶向PF噬菌体的治疗。至 为此，我们研制了针对PF噬菌体外壳蛋白的单抗和疫苗， COAB。我们的初步实验表明，抗体可以干扰生物膜的组织，促进抗生素的产生。 体外外显率。这表明通过给糖尿病患者接种疫苗来预防创面感染是可能的。 抗PF噬菌体的个体。然而，首先有必要在体内证明这些治疗方法的影响。 在目标2中，我们将测试针对PF噬菌体的抗体是否能促进小鼠模型的抗生素疗效。 PA生物被膜感染。 总而言之，这些目标代表了一种大胆而全新的方法来治疗PA生物被膜感染和慢性伤口 感染。如果成功，这些研究产生的数据将支持对Pf噬菌体作用的调查 伤口感染的病理生理学和针对PF噬菌体的新疗法。

Project Summary Pseudomonas aeruginosa (Pa) is a major human pathogen whose virulence is predicated upon its ability to form biofilms - slimy layers of polysaccharides and bacteria that allow Pa to colonize wounds and to evade antibiotics. My lab has recently uncovered novel roles for bacteriophages in the formation and function of Pa biofilms. We reported that Pf bacteriophages (Pf phages) produced by Pa spontaneously assemble the loose network of host and microbial polymers present at sites of Pa infection into a dense, highly stable, liquid crystal. This crystalline architecture enhances biofilm adhesiveness, viscosity, and resistance to desiccation. We also recently reported that Pf phage and this crystalline architecture contribute to antibiotic tolerance by binding and sequestering antibiotics. This effect is dependent on the charge properties of the antibiotic; positively charged antibiotics, like tobramycin, are efficiently sequestered within crystalline bundles of negatively charged phage while neutrally charged antibiotics, like ciprofloxacin, are not. These data suggest that presence of Pf phage may be an important factor in determining which antibiotics are effective against Pa. Together, these data suggest that Pa bacteria and Pf phage may partner in ways that contribute to the pathogenicity of biofilm infections. Consistent with this, our preliminary data suggest that, in a murine model of Pa biofilm wound infection, the presence of Pf phage contributes to chronic infection. These novel insights into the pathogenic contributions of Pf phage have the potential to open up a new front in the fight against Pa biofilms. However, first it is important to determine their relevance to human chronic wound infections. In Aim 1, we will assess whether Pa biofilms in human wounds have crystalline structure and define the relationship between Pf phage levels and clinical outcomes, including antibiotic resistance. Give these contributions to biofilm pathogensis, it may be beneficial to to target Pf phage therapeutically. To this end, we have developed monoclonal antibodies and vaccines directed against the Pf phage coat protein, CoaB. Our initial experiments suggest that antibodies can interfere with biofilm organization and promote antibiotic penetrance in vitro. This suggests that it may be possible to prevent Pa wound infections by immunizing diabetic individuals against Pf phage. However, first it is necessary to demonstrate the impact of these treatments in vivo. In Aim 2, we will test whether antibodies directed against Pf phage promote antibiotic efficacy in a mouse model of Pa biofilm infection. Together, these aims represent a bold and radically novel approach to Pa biofilm infections and chronic wound infections. If successful, the data generated from these studies will support investigations into the role of Pf phage in the pathophysiology of wound infections and novel therapies targeting Pf phage.