Mycobacteria Research Laboratory, Institute of Structural and Molecular Biology, School of Natural Sciences, Birkbeck, University of London, London, UK.
Methods Mol Biol. 2024;2833:35-42. doi: 10.1007/978-1-0716-3981-8_4.
Antimicrobial resistance (AMR) poses a serious threat to global health, potentially causing 10 million deaths per year globally by 2050. To tackle AMR, researchers from all around the world have generated a selection of various formulated (viz. nanoparticulate, liposomal) therapeutic combinations to be evaluated for new antimicrobial drug discovery. To meet the urgent need for accelerating new antibacterial drug development, we need rapid but reliable whole-cell assay methods and models to test formulated therapeutic combinations against several pathogens in different in vitro conditions as models of actual infections.Over the past two decades, high-throughput spot-culture growth inhibition assay (HT-SPOTi) has been demonstrated to be a gold-standard drug susceptibility method for evaluating novel chemotherapeutic entities and existing drugs against various microbes of global concern. Our modified HT-SPOTi method serves the purpose of evaluating drug combinations against Gram-positive/negative microorganisms as well as acid-fast bacilli. The newly developed and modified HT-SPOTi assay builds upon the limitations of our previously published method to incorporate antimicrobial susceptibility testing with formulated therapeutic combinations. The modified HT-SPOTi is compared with a range of other antimicrobial susceptibility testing methods and validated using a library of existing antibiotics as well as formulated therapeutic combinations. The modified HT-SPOTi assay can serve as an efficient and reliable high-throughput drug screening platform to discover new potential antimicrobial molecules, including as part of therapeutic formulations.This chapter describes the generation of drug susceptibility profile for formulated therapeutic combinations using modified HT-SPOTi in a semi-automated system.
抗菌药物耐药性（AMR）对全球健康构成严重威胁，到 2050 年，全球每年可能因此导致 1000 万人死亡。为了应对 AMR，来自世界各地的研究人员已经生成了一系列不同配方（即纳米颗粒、脂质体）的治疗组合，以评估新的抗菌药物发现。为了满足加速新型抗菌药物开发的迫切需求，我们需要快速但可靠的全细胞测定方法和模型，以在不同的体外条件下针对几种病原体测试配方治疗组合，作为实际感染的模型。在过去的二十年中，高通量点培养生长抑制测定（HT-SPOTi）已被证明是评估新型化疗实体和现有药物对全球关注的各种微生物的金标准药敏方法。我们修改的 HT-SPOTi 方法旨在评估针对革兰氏阳性/阴性微生物以及抗酸杆菌的药物组合。新开发和修改的 HT-SPOTi 测定方法基于我们之前发表的方法的局限性，将抗菌药物敏感性测试与配方治疗组合相结合。修改后的 HT-SPOTi 与一系列其他抗菌药物敏感性测试方法进行比较，并使用现有的抗生素库和配方治疗组合进行验证。修改后的 HT-SPOTi 测定方法可以作为一种高效可靠的高通量药物筛选平台，用于发现新的潜在抗菌分子，包括作为治疗配方的一部分。本章描述了使用修改后的 HT-SPOTi 在半自动系统中生成配方治疗组合的药物敏感性谱。
