i3S-Instituto de Investigação e Inovação e Saúde, Universidade do Porto, Porto, Portugal.
IBMC-Instituto de Biologia Celular e Molecular, Universidade do Porto, Porto, Portugal.
Microbiol Spectr. 2024 Oct 3;12(10):e0036224. doi: 10.1128/spectrum.00362-24. Epub 2024 Aug 27.
(Mab) is an emerging pathogen that poses a severe health threat, especially in people with cystic fibrosis and other chronic lung diseases. Available drugs are largely ineffective due to an exquisite intrinsic resistance, making Mab infections only comparable to multidrug-resistant tuberculosis. Current treatment is based on lengthy multidrug therapy, complicated by poor outcomes and high rates of treatment failure, recurrence, and mortality. Thus, finding new and more efficient drugs to combat this pathogen is urgent. However, drug discovery efforts targeting Mab have been limited, and traditional drug screening methods are labor-intensive, low-throughput, and do not reflect clinical effectiveness. Therefore, this work aimed to develop a new, efficient, and reliable tool for drug screening against Mab that can be used for identifying hits in a high-throughput manner and to select drug candidates for future clinical trials. We engineered two stable double-reporter strains of Mab capable of emitting strong fluorescent and luminescent signals. This is due to the expression of mScarlet protein and luciferase enzyme or the entire lux operon. Importantly, these strains maintain the same ground characteristics as the non-transformed Mab strain. We show that these new strains can be applied to various setups, from MIC determination in broth cultures and macrophage infection assays to infection (using the model). Using these strains enhances the potential for high-throughput screening of thousands of compounds in a fast and reliable way.
(Mab) is currently considered an "incurable nightmare." Its intrinsic resistance, high toxicity, long duration, and low cure rates of available therapies often lead to the clinical decision not to treat. Moreover, one of the significant drawbacks of anti-Mab drug development is the lack of correlation between susceptibility and clinical efficacy. Most drug screening assays are performed on Mab growing in liquid cultures. But being an intracellular pathogen, inducing granulomas and biofilm formation, the broth culture is far from ideal as drug-testing setup. This study presents new double-reporter Mab strains that allow direct real-time bacterial detection and quantification in a non-invasive way. These strains can be applied to an extensive range of experimental settings, far surpassing the utility of single-reporter bacteria. They can be used in all steps of the pre-clinical anti-Mab drug development pipeline, constituting a highly valuable tool to increase its success.
（Mab）是一种新兴病原体，对健康构成严重威胁，尤其是对囊性纤维化和其他慢性肺部疾病患者。由于其内在的高度耐药性，现有的药物在很大程度上无效，使得 Mab 感染的严重程度可与耐多药结核病相媲美。目前的治疗方法基于长期的多药物治疗，但治疗结果不理想，失败率、复发率和死亡率都很高。因此，迫切需要寻找新的、更有效的药物来对抗这种病原体。然而，针对 Mab 的药物发现工作一直受到限制，传统的药物筛选方法既费时费力，又通量低，且不能反映临床疗效。因此，这项工作旨在开发一种新的、高效的、可靠的 Mab 药物筛选工具，用于高通量筛选命中药物，并为未来的临床试验选择候选药物。我们构建了两种能够发出强荧光和发光信号的 Mab 稳定双报告菌株。这是由于 mScarlet 蛋白和荧光素酶或整个 lux 操纵子的表达。重要的是，这些菌株保持与非转化 Mab 菌株相同的地面特性。我们表明，这些新菌株可应用于各种设置，从肉汤培养物中的 MIC 测定和巨噬细胞感染测定到感染（使用 模型）。使用这些菌株可以增强在快速可靠的方式下对数千种化合物进行高通量筛选的潜力。
（Mab）目前被认为是一种“无法治愈的噩梦”。其内在的耐药性、高毒性、长疗程和现有治疗方法的低治愈率往往导致临床决定不进行治疗。此外，抗 Mab 药物开发的一个显著缺点是 药敏性与临床疗效之间缺乏相关性。大多数 Mab 药物筛选检测是在 Mab 在液体培养基中生长时进行的。但是，作为一种细胞内病原体，Mab 会诱导肉芽肿和生物膜形成，因此肉汤培养远非理想的 药物测试设置。本研究提出了新的双报告 Mab 菌株，可直接实时非侵入性检测和定量细菌。这些菌株可应用于广泛的实验设置，远远超过单报告细菌的用途。它们可用于 Mab 药物开发的临床前的各个阶段，是提高成功率的非常有价值的工具。
