Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra 400076, India.
ACS Infect Dis. 2024 Aug 9;10(8):2623-2636. doi: 10.1021/acsinfecdis.3c00605. Epub 2024 Jul 3.
Antibiotic resistance is a pressing health issue, with the emergence of resistance in bacteria outcompeting the discovery of novel drug candidates. While many studies have used Adaptive Laboratory Evolution (ALE) to understand the determinants of resistance, the influence of the drug dosing profile on the evolutionary trajectory remains understudied. In this study, we employed ALE on exposed to various concentrations of Norfloxacin using both cyclic constant and stepwise increasing drug dosages to examine their impact on the resistance mechanisms selected. Mutations in an efflux pump regulator, LfrR, were found in all of the evolved populations irrespective of the drug profile and population bottleneck, indicating a conserved efflux-based resistance mechanism. This mutation appeared early in the evolutionary trajectory, providing low-level resistance when present alone, with a further increase in resistance resulting from successive accumulation of other mutations. Notably, drug target mutations, similar to those observed in clinical isolates, were only seen above a threshold of greater than 4× the minimum inhibitory concentration (MIC). A combination of three mutations in the genes, R, MSMEG_1959, and MSMEG_5045, was conserved across multiple lineages, leading to high-level resistance and preceding the appearance of drug target mutations. Interestingly, in populations evolved from parental strains lacking the A efflux pump, the primary target of the R regulator, no R gene mutations are selected. Furthermore, evolutional trajectories originating from the ΔA strain displayed early arrest in some lineages and the absence of target gene mutations in those that evolved, albeit delayed. Thus, blocking or inhibiting the expression of efflux pumps can arrest or delay the fixation of drug target mutations, potentially limiting the maximum attainable resistance levels.
抗生素耐药性是一个紧迫的健康问题，细菌耐药性的出现已经超过了新型药物候选物的发现。虽然许多研究已经使用适应性实验室进化（ALE）来了解耐药性的决定因素，但药物剂量方案对进化轨迹的影响仍研究不足。在这项研究中，我们使用 ALE 对暴露于不同浓度诺氟沙星的细菌进行了研究，使用循环恒定和逐步增加的药物剂量来研究它们对选择的耐药机制的影响。在所有进化种群中，无论药物谱和种群瓶颈如何，都发现了外排泵调节剂 LfrR 的突变，表明存在保守的基于外排的耐药机制。这种突变出现在进化轨迹的早期，单独存在时提供低水平的耐药性，随后其他突变的连续积累导致耐药性进一步增加。值得注意的是，只有在药物浓度大于最小抑菌浓度（MIC）的 4 倍以上时，才会出现与临床分离株中观察到的类似的药物靶标突变。在多个谱系中，基因 R、MSMEG_1959 和 MSMEG_5045 中的三个突变组合是保守的，导致高水平耐药性，并在药物靶标突变出现之前。有趣的是，在缺乏 A 外排泵的亲本菌株进化而来的种群中，R 调节剂的主要靶标 A efflux pump 没有选择 R 基因突变。此外，来自ΔA 菌株的进化轨迹在一些谱系中早期停滞，并且在进化的谱系中没有靶基因突变，尽管延迟。因此，阻断或抑制外排泵的表达可以阻止或延迟药物靶标突变的固定，从而限制可达到的最大耐药水平。
