Tuberculosis continues to kill millions of people each year. The main difficulty in eradication of the disease is the prolonged duration of treatment, which takes at least 6 months. Persister cells have long been associated with failed treatment and disease relapse because of their phenotypical, though transient, tolerance to drugs. By targeting these persisters, the duration of treatment could be shortened, leading to improved tuberculosis treatment and a reduction in transmission. The unique in vivo environment drives the generation of persisters; however, appropriate in vivo mycobacterial persister models enabling optimized drug screening are lacking. To set up a persister infection model that is suitable for this, we infected zebrafish embryos with in vitro-starved Mycobacterium marinum. In vitro starvation resulted in a persister-like phenotype with the accumulation of stored neutral lipids and concomitant increased tolerance to ethambutol. However, these starved wild-type M. marinum organisms rapidly lost their persister phenotype in vivo. To prolong the persister phenotype in vivo, we subsequently generated and analyzed mutants lacking functional resuscitation-promoting factors (Rpfs). Interestingly, the ΔrpfAB mutant, lacking two Rpfs, established an infection in vivo, whereas a nutrient-starved ΔrpfAB mutant did maintain its persister phenotype in vivo. This mutant was, after nutrient starvation, also tolerant to ethambutol treatment in vivo, as would be expected for persisters. We propose that this zebrafish embryo model with ΔrpfAB mutant bacteria is a valuable addition for drug screening purposes and specifically screens to target mycobacterial persisters.

中文翻译：

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斑马鱼胚胎模型，用于评估分枝杆菌药的药效。

结核病每年继续杀死数百万人。根除该疾病的主要困难是治疗时间延长，至少需要6个月。持久性细胞长期以来一直与失败的治疗和疾病复发相关，因为它们具有表型（尽管是短暂的）对药物的耐受性。通过针对这些坚持者，可以缩短治疗时间，从而改善结核病治疗并减少传播。独特的体内环境推动了持久性的产生。然而，缺乏合适的体内分枝杆菌持续性模型，可以优化药物筛选。为了建立适合这种情况的持久性感染模型，我们在体外感染了斑马鱼胚胎饥饿的海洋分枝杆菌。体外饥饿导致持久性样表型，其中存储了中性脂质，并且对乙胺丁醇的耐受性增加。然而，这些饥饿的野生型海藻分支杆菌生物体在体内迅速丧失了其持久性表型。为了延长体内的持久性表型，我们随后产生并分析了缺乏功能性复苏促进因子（Rpfs）的突变体。有趣的是，缺乏两个Rpfs的ΔrpfAB突变体在体内建立了感染，而营养缺乏的ΔrpfAB突变体在体内确实维持了其持久性表型。。在营养饥饿后，该突变体还耐受体内乙胺丁醇的治疗，这对于持久性药物是可以预期的。我们建议，具有ΔrpfAB突变细菌的斑马鱼胚胎模型是用于药物筛选的有价值的补充，特别是针对目标分枝杆菌持久性的筛选。