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

项目概要/摘要 肺恶化是囊性纤维化（CF）最常见的表现之一， 影响了3万美国人的多系统疾病这些恶化通常是由高药物引起的- 耐药革兰氏阴性菌，定植于CF患者的肺部。在这些生物中， 洋葱伯克霍尔德氏菌复合体（Burkholderia cepacia complex，BCC）BCC具有广泛的内在抗生素耐药性， 在抗生素治疗期间的选择压力下获得进一步的耐药机制;泛耐药菌株 可以在反复使用抗生素后出现。BCC感染与频繁住院有关， CF患者的死亡率增加，但目前还没有新的抗生素对BCC具有活性， 发展管道和替代治疗战略是迫切需要的。建议的目标 研究是鉴定已知的单独或组合具有抗BCC活性的生物活性化合物 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种已知的生物活性物质， 小分子，包括所有FDA批准的药物，无论是单独使用还是与六种抗生素联合使用， 用于治疗基底细胞癌（美罗培南、头孢他啶、米诺环素、左氧氟沙星、甲氧苄啶-磺胺甲恶唑， 妥布霉素，各自处于单独的无效浓度），以鉴定化合物和化合物/抗菌剂 抑制代表性BCC分离物生长的组合。在目标2中，我们将进一步描述 在使用几种互补的体外模型筛选中鉴定出的有前途的化合物。一是 对化合物和组合进行活性谱和剂量反应测试， 使用喷墨打印机辅助棋盘阵列协同研究以及时间杀灭协同研究的30个BCC分离株 问题研究我们还将评估化合物在治疗期间防止抗生素耐药性出现的能力。 治疗然后，为了更好地近似细菌在患有肺结核的人的肺中生活的环境， CF，其特征在于粘度增加，粘蛋白、白蛋白、氨基酸和游离氨基酸浓度高， DNA，以及相对于标准体外抗菌药物敏感性测试条件的较低氧分压，我们将 在微需氧环境中使用人工痰培养基测定测试化合物和组合。与此 我们将评估是否在更接近于以下情况的条件下保持活动： 它们将用于临床实践。当项目完成后，我们希望已经确定了一个集合， 的分子与以前未认识到的活性对BCC，并已确定其中哪些是最重要的 可能在CF患者中临床有效。鉴定具有以下特征的充分表征的化合物： 对BCC的潜在治疗活性将有助于未来在动物模型和人体试验中的评估， 为了开发迫切需要的新的治疗选择BCC。

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.