Kirubakar Greana, Johnston Luana, Nae Rin Lee Bom, Russell David G, Simwela Nelson V
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA.
Antimicrob Agents Chemother. 2025 Oct;69(10):e0079525. doi: 10.1128/aac.00795-25. Epub 2025 Sep 11.
Development of new and improved tuberculosis (TB) chemotherapies is hampered by antibiotic resistance and drug tolerance by (). Phenotypic drug tolerance, a phenomenon where populations can temporarily survive therapeutic antibiotic concentrations, represents a significant hurdle to TB treatment and is indeed one of the factors responsible for prolonged TB therapy. Assays that can identify compounds with improved efficacy against drug-tolerant are urgently required to improve TB treatment regimens. Here, we report the development of a 96-well plate assay capable of identifying anti- drugs with activity against drug-tolerant in physiologically relevant intracellular environments within macrophages. Primary murine macrophages, modified either by immunological activation or specific CRISPR/Cas9 gene knockouts to generate tolerance-inducing environments, were infected with an strain constitutively expressing luciferase. Following drug exposure, differences in bacterial survival were measured by bacterial outgrowth after lysis of the host macrophages. By monitoring luciferase in infected macrophages before, during, and after drug treatment, we confirmed earlier observations that host immune stresses trigger induction of drug tolerance. However, while host stresses induced tolerance against some anti-TB compounds, the same host stresses were synergistic with other anti-TB drugs. Our assay provides the ability to profile the activities of anti-TB drugs on bacteria in intracellular host environments, which is critical to the rational design of drug combinations that provide optimal coverage of the sub-populations in the infected host.
新型和改良的结核病（TB）化疗药物的研发受到抗生素耐药性和（）药物耐受性的阻碍。表型药物耐受性是指细菌群体能够在治疗性抗生素浓度下暂时存活的现象，这是结核病治疗的一个重大障碍，也是导致结核病治疗疗程延长的因素之一。迫切需要能够识别对耐药结核杆菌疗效更佳的化合物的检测方法，以改进结核病治疗方案。在此，我们报告了一种96孔板检测方法的开发，该方法能够在巨噬细胞内生理相关的细胞内环境中识别对耐药结核杆菌具有活性的抗结核药物。通过免疫激活或特定的CRISPR/Cas9基因敲除进行修饰以产生耐受性诱导环境的原代小鼠巨噬细胞，被组成性表达荧光素酶的结核杆菌菌株感染。药物暴露后，通过宿主巨噬细胞裂解后的细菌生长来测量细菌存活的差异。通过监测药物治疗前、治疗期间和治疗后感染巨噬细胞中的荧光素酶，我们证实了早期的观察结果，即宿主免疫应激会触发药物耐受性的诱导。然而，虽然宿主应激诱导了对某些抗结核化合物的耐受性，但相同的宿主应激与其他抗结核药物具有协同作用。我们的检测方法能够分析抗结核药物在细胞内宿主环境中对细菌的活性，这对于合理设计能够最佳覆盖感染宿主中结核杆菌亚群的药物组合至关重要。
