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。一种选择是通过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，该项目探索了其他进化假说，以提高治疗效果，使用实验室进化实验、全基因组序列分析和功能遗传分析的组合，所有这些都与模式病原体铜绿假单胞菌一起。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(即序贯抗生素疗法)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存在不同的随后应用的抗生素时造成不利(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的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，所获得的成果将用于进一步解决首要目标，即加强我们对细菌对时间波动的抗生素环境的适应的了解。更具体地说，将进一步分析第一个供资阶段最耐人寻味的两个结果，以更全面地了解已发现的现象，包括(I)负滞后是提高序贯疗法疗效的关键原则，以及(Ii)三种β-内酰胺类抗生素序贯疗法的意外高疗效。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负滞后的一般发生情况，并评估负滞后现象在多大程度上具有限制细菌进化速度的一般潜力。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的冷冻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。综上所述，该项目将产生一个关于环境条件、选择动态和导致细菌适应波动环境的相关基因变化的全面数据库。因此，这个数据库可以帮助设计新的可持续的抗生素疗法，利用进化知识来实现消除感染剂和最大限度地减少耐药性传播。

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.