Identification of potentiators of antimicrobial activity against multidrug-resistant Burkholderia cepacia complex infections in cystic fibrosis

项目摘要/摘要 肺恶化是囊性纤维化最常见的表现之一，囊性纤维化是一种限制生命的疾病， 影响3万美国人的多系统疾病。这些恶化通常是由高度药物- 耐药的革兰氏阴性菌，定植于CF患者的肺部。在这些生物中，最多的是 令人担忧的是洋葱伯克霍尔德氏菌复合体(BCC)。BCC具有广泛的内在抗生素耐药性，很容易 在抗生素治疗期间在选择性压力下获得进一步的耐药机制；泛耐药菌株 可在反复使用抗生素疗程后出现。基底细胞癌感染与频繁住院和 增加了CF患者的死亡率，但在美国没有新的具有抗BCC活性的抗生素 迫切需要发展管道和替代治疗战略。建议的目标是 研究是确定已知的生物活性化合物，这些化合物单独或联合具有抗BCC的活性 用抗生素。这项工作将通过两个目标进行。在目标1中，我们将测试14,000种已知的生物活性 小分子药物，包括所有FDA批准的药物，通常单独或与六种抗生素联合使用 用于治疗BCC(美罗培南、头孢他啶、米诺环素、左氧氟沙星、甲氧嘧啶-磺胺甲恶唑)和 妥布霉素，每个都处于单独无效的浓度)，以识别化合物和化合物/抗菌药物 抑制典型BCC分离株生长的组合。在目标2中，我们将进一步描述活动的特征 使用几个互补的体外模型在筛选中确定的有前景的化合物。首先，我们将 对化合物及其组合进行活性光谱和剂量反应测试 利用喷墨打印机辅助棋盘阵列协同作用研究30株BCC分离物以及消时增效作用 学习。我们还将评估化合物防止抗生素耐药性出现的能力 治疗。然后，为了更好地接近细菌在肺部的生存环境， Cf，其特征是粘度增加，粘蛋白、白蛋白、氨基酸和游离的浓度较高 DNA，以及较低的氧分压相对于标准的体外药敏试验条件，我们将 在微需氧环境中使用人工痰介质测试化合物和化合物。有了这个 方法，我们将评估活动是否保持在更接近以下条件的条件下 它们将用于临床实践。当项目完成时，我们希望已经确定了一个集合 具有以前未知的抗BCC活性的分子，并已确定其中哪些是最 可能在临床上对CF患者有效。具有良好特性的化合物的鉴定 针对基底细胞癌的潜在治疗活性将有助于未来在动物模型和人体试验中进行评估 以开发迫切需要的新的基底细胞癌治疗方案。

Project Summary/Abstract Pulmonary exacerbations are one of the most common manifestations of cystic fibrosis (CF), a life-limiting, multisystem disease that affects 30,000 Americans. These exacerbations are often caused by highly drug- resistant Gram-negative bacteria that colonize the lungs of people with CF. Among such organisms, the most feared is the Burkholderia cepacia complex (Bcc). Bcc have extensive intrinsic antibiotic resistance and readily acquire further resistance mechanisms under selective pressure during antibiotic treatment; pan-resistant strains can emerge following repeated antibiotic courses. Bcc infection is associated with frequent hospitalizations and increased mortality in people with CF, but there are no new antibiotics with activity against BCC in the development pipeline, and alternative treatment strategies are urgently needed. The goal of the proposed research is to identify known bioactive compounds that have activity against Bcc either alone or in combination with antibiotics. This work will be carried out through two aims. In Aim 1, we will test 14,000 known bioactive small molecules, including all FDA-approved drugs, both alone and in combination with six antibiotics commonly used to treat Bcc (meropenem, ceftazidime, minocycline, levofloxacin, trimethoprim-sulfamethoxazole, and tobramycin, each at an individually ineffective concentration), to identify compounds and compound/antibacterial combinations that inhibit growth of a representative Bcc isolate. In Aim 2, we will further characterize the activity of promising compounds identified in the screen using several complementary in vitro models. First, we will perform spectrum-of-activity and dose-response testing of compounds and combinations against a collection of 30 Bcc isolates using inkjet printer-assisted checkerboard array synergy studies as well as time-kill synergy studies. We will also evaluate the capacity of compounds to prevent the emergence of antibiotic resistance during treatment. Then, in order to better approximate the environment in which bacteria live in the lungs of people with CF, which is characterized by increased viscosity, high concentrations of mucin, albumin, amino acids, and free DNA, and lower oxygen tension relative to standard in vitro antimicrobial susceptibility testing conditions, we will test compounds and combinations using an artificial sputum medium in a microaerophilic environment. With this approach, we will assess whether activity is maintained under conditions more closely resembling those in which they would be used in clinical practice. When the project is completed, we expect to have identified a collection of molecules with previously unrecognized activity against Bcc and to have determined which of these are most likely to be clinically effective in people with CF. Identification of well-characterized compounds that have potential therapeutic activity against Bcc will facilitate future evaluation in animal models and human trials in order to develop desperately needed new therapeutic options for Bcc.