ABSTRACT

The bacterial populations surviving in the presence of antibiotics contain cells that have gained genetic resistance, phenotypic resistance and tolerance to antibiotics. Isolation of live bacterial population, surviving against antibiotics, from the milieu of high proportions of dead/damaged cells will facilitate the study of the cellular/molecular processes used by them for survival. Here we present a Percoll gradient centrifugation based method for the isolation of enriched population of Mycobacterium smegmatis surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin. From the time of harvest, throughout the enrichment and isolation processes, and up to the lysis of the cells for total RNA preparation, we maintained the cells in the presence of the antibiotic to avoid changes in their metabolic status. The total RNA extracted from the enriched population of live antibiotic-surviving population showed structural integrity and purity. We analysed the transcriptome profile of the antibiotic-surviving population and compared it with the orthologue genes of Mycobacterium tuberculosis that conferred antibiotic tolerance on tubercle bacilli isolated from the tuberculosis patients under treatment with four antitubercular antibiotics. Statistically significant comparability between the gene expression profiles of the antibiotic tolerance associated genes of M. smegmatis and M. tuberculosis validated the reliability/utility of the method.

中文翻译：

---

一种富集、分离和验证在杀菌浓度的利福平和莫西沙星存在下存活的耻垢分枝杆菌群体的方法

 抽象的

在抗生素存在下存活的细菌群体含有获得遗传抗性、表型抗性和抗生素耐受性的细胞。从高比例死亡/受损细胞的环境中分离出能够抵抗抗生素生存的活细菌群，将有助于研究它们赖以生存的细胞/分子过程。在这里，我们提出了一种基于 Percoll 梯度离心的方法，用于分离在杀菌浓度的利福平和莫西沙星存在下存活的耻垢分枝杆菌富集群。从收获时，整个富集和分离过程，一直到裂解细胞以制备总RNA，我们将细胞维持在抗生素存在下，以避免其代谢状态发生变化。从活抗生素存活群体富集群体中提取的总 RNA 显示出结构完整性和纯度。我们分析了抗生素存活群体的转录组谱，并将其与结核分枝杆菌的直系同源基因进行比较，该基因赋予从接受四种抗结核抗生素治疗的结核病患者中分离出的结核杆菌抗生素耐受性。耻垢分枝杆菌和结核分枝杆菌的抗生素耐受性相关基因的基因表达谱之间具有统计学上显着的可比性，验证了该方法的可靠性/实用性。