The role of Staphylococcus aureus aggregate formation in establishing biofilms in chronic orthopaedic periprosthetic joint infection.

项目总结 人工关节置入后的假体周围关节感染(PJI)是一种毁灭性的并发症 病人和外科医生。感染通常需要一个艰难的反复手术过程，需要多个疗程 通过全身或局部使用抗生素骨水泥给药的广谱抗生素。在许多 即使分离出的病原体对注射的药物敏感，感染也不能消除 抗生素，截肢并不少见。令人震惊的是，最近的一项研究报告称，26%的患者接受了 PJI在5年内死亡，是因感染和其他原因被替换的人数的两倍 存活率低于卧床不起、黑色素瘤和乳腺癌的患者。虽然感染的发生率 在全髋关节和全膝关节置换术中，手术次数相对较少(分别为2.0%和2.4% 预计将呈指数级增长，预计到2020年，美国将有15,000个髋关节和50,000个膝盖PJI。生物膜 细菌病原体在种植体组件上的形成与慢性PJI的难治性有关。 生物膜是生活在胞外聚合物(Eps)内的细菌细胞附着群落。 由细菌聚合物和宿主成分组成的基质。生物膜的形成是一种用于 它对抗生素有很高的耐受性，对宿主免疫也有抵抗力。然而， 重要的临床悬而未决的问题仍然存在，即生物膜是如何从推测的 在现代OR和AND高度受控的环境中，少量细胞进入手术部位 感染关节中的生物膜在哪里生长？葡萄球菌的聚集(或血液中的凝集)是一种已知的 脓毒症中的毒力因子但最近研究表明，在人类滑膜中迅速形成的聚集体 液体可以附着在表面形成生物膜。聚集还提供了类似生物膜的快速耐受性，这可能 在细菌附着到植入物之前保护它们，并建立成熟的生物膜。而体外模型则专注于 与单个细胞的相互作用几乎对细胞的附着和生长动力学一无所知 集合体。在目标1中，我们将测定牛关节滑液中的聚集动力学，以确定 对抗生素的耐受性和中性粒细胞的摄取需要大量的聚集体。我们亦会评估 宿主因子、细菌黏附蛋白和EPS组分对聚集动力学的相对贡献 以及使用表面活性剂作为外科冲洗剂，以防止和分散聚集体。在目标2中，我们将量化 骨料在不同骨科材料上的附着率和后续生物膜的发展 流动细胞和恒化器模型中的受控剪应力。在目标3中，我们将使用一种新的琼脂包装 从原位生长出的整个种植体上定位和量化生物膜的培养方法 表面。受感染的重建关节具有复杂的材料和机械力分布，并且 理解这如何导致细胞聚集体和随后的附着的优先利基 生物膜的形成对于外科医生和知情的种植体设计是非常感兴趣的。

PROJECT SUMMARY Periprosthetic joint infection (PJI) after placement of an artificial joint is a devastating complication for both the patient and surgeon. Infection typically requires an arduous course of repeated surgeries with multiple courses of broad-spectrum antibiotics administered systemically or locally by antibiotic loaded bone cements. In many cases the infection cannot be resolved, even though the isolated pathogen is sensitive to the administered antibiotics, and amputation is not uncommon. Alarmingly a recent study reports that 26% of patients treated for PJI were dead within 5 years, twice the number of those being replaced for reasons other than infection and survivorship rates less than those with prostrate, melanoma and breast cancers. Although the infection incidence in total hip and total knee arthroplasties is relatively low (2.0% and 2.4% respectively), the number of procedures is forecast to grow exponentially and 15,000 hips and 50,000 knee PJIs are predicted in the US by 2020. Biofilm formation on the implant components by bacterial pathogens is associated with the intractability of chronic PJIs. Biofilms are attached communities of bacterial cells living within an extracellular polymeric substances (EPS) matrix composed of bacterial polymers and host components. Biofilm formation is a mechanism used for protection, and it confers high levels of tolerance to antibiotics and resistance to host immunity. However important unanswered clinical questions remain, how do biofilms become established from what is presumably a small numbers of cells entering the surgical site in the highly controlled environment of the modern OR and where do biofilms reside in the infected joint? Aggregation (or agglutination in blood) in staphylococci is a known virulence factor in sepsis but recently it has been shown that aggregates which form rapidly in human synovial fluid can attach to surfaces to form biofilms. Aggregation also affords rapid biofilm-like tolerance which may protect the bacteria before they attach to implants and establish mature biofilms. While in vitro models focus on interaction with single cells almost nothing is known about the kinetics of attachment and growth from aggregates. In Aim 1 we will determine the kinetics of aggregation in bovine synovial fluid to determine what size of aggregates are required to confer tolerance to antibiotics and neutrophil uptake. We will also assess the relative contribution of host factors, bacterial adhesion proteins and EPS components on aggregation kinetics and the use of surfactants as surgical irrigants to prevent and disperse aggregates. In Aim 2 we will quantify the attachment rate of aggregates and subsequent biofilm development on different orthopaedic materials under controlled shear stresses in flow-cell and chemostat models. In Aim 3 we will use a novel agar encasement culturing method to map and quantify biofilms to specific locations on entire implants from in situ outgrowth from the surface. An infected reconstructed joint has a complex distribution of materials and mechanical forces, and understanding how this leads to preferential niches for the attachment of cellular aggregates and subsequent biofilm formation is of profound interest for surgeons and for informed implant design.