Mechanisms of antibiotic failure during osteomyelitis

项目摘要/摘要 骨髓炎是一种侵袭性骨感染，最常见的致病菌是细菌。 金黄色葡萄球菌。骨髓炎的成功治疗需要长期的抗生素治疗以及 侵入性外科手术切除感染的和不能存活的骨头。尽管采取了这些激进的措施，但几乎 每5名骨髓炎患者中就有1名治疗失败。这包括接受抗生素治疗的患者，这些患者已被证实 体外抗病原菌活性。这些观察将骨髓炎定位为 治疗-顽固性感染。这项提案的目标是定义有助于 骨髓炎期间抗生素失效。我们假设细菌毒力因子和宿主反应 促进体内细菌的物理屏蔽，有助于抗生素耐受。在成功完成后 在拟议的实验中，我们希望能够确定抗生素失效的有针对性的机制 骨髓炎，最终目标是最大限度地提高治疗效果，最大限度减少并发症，并创造 检测和对抗人类抗生素耐受性的新方法。 在使用我们建立的小鼠骨髓炎模型进行的初步研究中，我们证明了S. 金黄色葡萄球菌在体内迅速产生对抗生素杀灭的耐受性。组织学分析揭示了一个特征 感染骨骼中细菌群落周围的“假包囊”。假囊的形成被认为是 涉及关键的毒力因子，如凝固酶和凝集素，它们共同赋予金黄色葡萄球菌以其 凝血的独特能力。我们假设这些毒力因子与抗生素耐受性有关 在骨髓炎期间，通过物理屏蔽宿主组织内的细菌。目标1将严格检验这一假设， 同时还使用无偏见的体内细菌筛选来识别抗生素耐受性的新决定因素。我们 将使用创新的金黄色葡萄球菌报告菌株跟踪抗生素和细菌的相对定位 再加上荧光抗生素。目标2将检验互补性假设：规范的宿主凝血 有助于保护细菌和抗生素耐受性。这一目标还将采用我们的新工作流程 骨骼的成像质谱学(IMS)，这将使基于发现的宿主分析物分析成为可能 体内的抗生素屏障。我们还将利用IMS作为一种无标签跟踪感染患者抗生素的方式 纸巾。Aim 3将使用成对的细菌分离株和骨髓炎患者的血清样本来测试 在生理相关介质中的生长改变了抗生素的杀伤力，并将体外抗生素耐受性与关键 临床参数。总的来说，本提案中的实验将定义导致抗生素的机制 骨髓炎期间的耐受性，为改善抗生素治疗的新干预铺平了道路，限制 治疗失败，减少这种使人衰弱的疾病的发病率。

PROJECT SUMMARY / ABSTRACT Osteomyelitis is an invasive infection of bone most commonly caused by the bacterial pathogen Staphylococcus aureus. Successful treatment of osteomyelitis requires prolonged antibiotic treatment as well as invasive surgical procedures to remove infected and nonviable bone. Despite these aggressive measures, nearly 1 in 5 patients with osteomyelitis will fail treatment. This includes patients who receive antibiotics with proven in vitro activity against the causative pathogen. These observations position osteomyelitis as a paradigm for treatment-recalcitrant infection. The goal of this proposal is to define bacterial and host factors that contribute to antibiotic failure during osteomyelitis. We hypothesize that bacterial virulence factors and host responses that promote physical shielding of bacteria in vivo contribute to antibiotic tolerance. Upon successful completion of the proposed experiments, we expect to have defined targetable mechanisms underlying antibiotic failure during osteomyelitis, with the ultimate goal of maximizing treatment efficacy, minimizing complications, and creating new approaches to detect and combat antibiotic tolerance in humans. In preliminary studies using our established murine model of osteomyelitis, we demonstrate that S. aureus rapidly develops tolerance to antibiotic killing in vivo. Histologic analyses reveal a characteristic “pseudocapsule” surrounding bacterial communities in infected bone. Pseudocapsule formation is postulated to involve key virulence factors known as coagulases and agglutinins, which together endow S. aureus with its distinctive ability to coagulate blood. We hypothesize that these virulence factors contribute to antibiotic tolerance during osteomyelitis by physically shielding bacteria within host tissues. Aim 1 will rigorously test this hypothesis, while also identifying novel determinants of antibiotic tolerance using an unbiased, in vivo bacterial screen. We will track the relative localization of antibiotics and bacteria using innovative new S. aureus reporter strains coupled with fluorescent antibiotics. Aim 2 will test the complementary hypothesis that canonical host coagulation contributes to shielding of bacteria and antibiotic tolerance. This Aim will also employ our new workflow for imaging mass spectrometry (IMS) of bone, which will enable discovery-based profiling of host analytes that form antibiotic barriers in vivo. We will also leverage IMS as a modality for label-free tracking of antibiotics in infected tissues. Aim 3 will use paired bacterial isolates and serum samples from patients with osteomyelitis to test how growth in a physiologically relevant medium alters antibiotic killing, and to link in vitro antibiotic tolerance to key clinical parameters. Collectively, the experiments in this proposal will define mechanisms leading to antibiotic tolerance during osteomyelitis, paving the way for new interventions that improve antibiotic therapy, limit treatment failure, and reduce the morbidity of this debilitating disease.