Chengan Kameshwari, Hind Charlotte, Stanley Maria, Wand Matthew E, Nagappa Lakshmeesha K, Howland Kevin, Hanson Tanith, Martín-Escolano Rubén, Tsaousis Anastasios D, Bengoechea José A, Mark Sutton J, Smales Christopher M, Moore Simon J
School of Biosciences, Division of Natural Sciences, University of Kent, Canterbury, CT7 2NJ, United Kingdom.
School of Biological and Behavioural Sciences, Queen Mary University of London, London, E1 4NS, United Kingdom.
NPJ Antimicrob Resist. 2023 Dec 18;1(1):16. doi: 10.1038/s44259-023-00018-z.
Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease-causing microbes. To provide a host-specific tool to study antibiotic susceptibility and resistance, here we develop Klebsiella pneumoniae cell-free gene expression (CFE) systems from laboratory and clinical isolates. Using proteomics, we identify relative differences and unique proteins for these new CFE systems in comparison to an Escherichia coli MG1655 CFE model. Then we profile antimicrobial susceptibility in parallel with whole cells to quantify CFE antibiotic potency. Finally, we apply this native CFE tool to study AMR variants at a proof-of-concept level. Definably we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin-a genotype observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. Overall, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial extract processing requires Biosafety Level 2, the CFE system is non-living, suitable for long-term storage and study in a Biosafety Level 1 lab. We anticipate the K. pneumoniae CFE bioassay is advantageous for host-specific antimicrobial testing, the characterisation of intracellular AMR variants and potentially structure-activity relationship studies.
抗菌药物耐药性（AMR）是一种在多种致病微生物中传播的全球性问题。为了提供一种针对宿主的工具来研究抗生素敏感性和耐药性，我们在此开发了来自实验室和临床分离株的肺炎克雷伯菌无细胞基因表达（CFE）系统。通过蛋白质组学，我们确定了这些新的CFE系统与大肠杆菌MG1655 CFE模型相比的相对差异和独特蛋白质。然后，我们与全细胞并行分析抗菌药物敏感性，以量化CFE抗生素效力。最后，我们将这种原生CFE工具应用于概念验证水平的AMR变体研究。我们明确表明，RpoB H526L使CFE对利福平的耐药性增加了58倍——这是在耐利福平结核分枝杆菌临床分离株中观察到的一种基因型。总体而言，我们提供了一种无细胞合成生物学策略，用于分析肺炎克雷伯菌的抗生素敏感性和耐药性。虽然初始提取物处理需要生物安全2级，但CFE系统是无生命的，适合在生物安全1级实验室中长期储存和研究。我们预计肺炎克雷伯菌CFE生物测定法对于宿主特异性抗菌测试、细胞内AMR变体的表征以及潜在的构效关系研究具有优势。
