Evolutionary adaptation of bacteria to changing antibiotic environments

抗生素耐药性已成为全球健康的主要威胁。迫切需要治疗细菌感染的新选择。一个选择是利用我们对细菌进化的理解来改进治疗。在第一个资助阶段，本项目利用实验室进化实验、全基因组序列分析和功能遗传分析相结合，以铜绿假单胞菌为模型病原体，探索了不同的进化假设，以提高治疗效果。最重要的发现之一是，细菌的适应能力可以通过改变抗生素环境（即，序贯抗生素治疗）来限制，特别是如果(i)抗生素快速交替（例如，每12小时），（ii）抗生素诱导的细菌生理变化在不同的抗生素存在下导致不利，随后应用的抗生素（即，负滞后），和/或（iii）由于进化权衡（即进化的附带敏感性），对一种抗生素的耐药性进化导致对第二种抗生素的敏感性增加。在拟议的第二个资助阶段，获得的结果将用于进一步解决总体目标，以提高我们对细菌适应暂时波动的抗生素环境的理解。更具体地说，将进一步分析第一阶段资助的两个最有趣的结果，以便更全面地了解所发现的现象，包括(i)负迟滞作为增强顺序治疗疗效的关键原则，以及（ii）使用三种β -内酰胺类抗生素进行顺序治疗的意想不到的高疗效。在前一种情况下，该项目将评估不同抗生素、不同确切治疗条件以及铜绿假单胞菌基因型中阴性迟滞现象的普遍发生情况，并评估阴性迟滞现象在多大程度上具有限制细菌进化速率的普遍潜力。在第二种情况下，将结合基因组学和功能遗传学对先前进行的进化实验中冷冻保存的细菌进行详细的表型表征，以了解三种相关抗生素的交替如何限制细菌的适应。总的来说，这个项目将产生一个关于环境条件、选择动力学和细菌适应波动环境所涉及的遗传变化的综合数据库。因此，该数据库可能有助于设计新的可持续抗生素疗法，利用进化知识来实现消除感染因子和最小化耐药性传播。

Antibiotic resistance has become a major threat to global health. New options for treatment of bacterial infections are urgently required. One option is to improve therapy by exploiting our understanding of bacterial evolution. During the first funding phase, this project explored alternative evolutionary hypotheses to improve treatment efficacy using a combination of evolution experiments in the laboratory, whole genome sequence analysis, and functional genetic analysis, all with the model pathogen Pseudomonas aeruginosa. One of the most important findings is that the ability of bacteria to adapt can be constrained through changing antibiotic environments (i.e., sequential antibiotic therapy), especially if (i) antibiotics are alternated rapidly (e.g., every 12 hours), (ii) antibiotic-induced physiological changes in the bacteria cause disadvantages in the presence of a different, subsequently applied antibiotic (i.e., negative hysteresis), and/or (iii) evolution of resistance to one antibiotic causes increased sensitivity to a second antibiotic due to an evolutionary trade-off (i.e., evolved collateral sensitivity). During the proposed second funding phase, the obtained results will be used to further address the overarching objective to enhance our understanding of bacterial adaptation to temporally fluctuating antibiotic environments. More specifically, two of the most intriguing results from the first funding phase will be further analysed, in order to obtain a more complete understanding of the discovered phenomena, including (i) negative hysteresis as a key principle to enhance efficacy of sequential therapy and (ii) unexpected high efficacy of sequential treatments with three beta-lactam antibiotics. In the former case, the project will assess the general occurrence of negative hysteresis across different antibiotics, different exact treatment conditions, and also P. aeruginosa genotypes and evaluate to what extent the phenomenon of negative hysteresis has general potential to constrain bacterial evolutionary rates. In the second case, cryopreserved bacteria from the previously performed evolution experiment will be characterized in detail phenotypically in combination with genomics and functional genetics, in order to understand how the alternation of three related antibiotics can constrain bacterial adaptation. Taken together, this project will yield a comprehensive database on the environmental conditions, the selective dynamics, and the involved genetic changes that underlie bacterial adaptation to fluctuating environments. This database may thereby help the design of new sustainable antibiotic therapy that exploits evolutionary knowledge to achieve both eliminating the infectious agent and minimizing resistance spread.