Determining the architecture of antibiotic resistance evolvability

抗生素耐药性日益普遍是公共卫生和农业的重大危机。细菌进化出耐药性的能力可能会发生巨大变化，但我们预测哪些细菌会继续进化出高水平耐药性的能力目前是有限的。了解是什么促成了耐药性的进化潜力，将使我们能够开发新的干预措施来抑制抗菌素耐药性。因此，了解促进耐药性“可进化性”的基因组机制是很重要的。我们将研究基因组多样性如何促进耐药性的进化。与其他专注于单一细菌物种的工作不同，我们将研究物种间基因组多样性如何促进进化抗性的能力。我们将专注于假单胞菌，一个令人难以置信的多样化属，包括环境，共生和致病生物。假单胞菌每年给全球卫生和农业部门造成超过1500亿英镑的经济负担。假单胞菌的抗生素耐药性正在迅速增加，了解是什么使耐药性演变为重要的抗假单胞菌抗生素是维持管理假单胞菌能力的关键。因此，我们将确定基因组水平的变异如何促进抗假单胞菌抗生素耐药性的进化，包括最近上市的一种专门针对假单胞菌生理学的抗生素（cefiderocol）。来自假单胞菌的证据表明，即使看似微小的基因组内容差异也可能对进化出耐药性的可能性产生广泛的影响。先前的研究表明，一个单一的“可进化性基因”可以影响假单胞菌是否能够进化出对抗生素头孢他啶的高水平耐药性。然而，我们目前不知道这种机制一般运作的程度。具体而言，对于(1)不同抗生素类别的耐药性进化性如何变化，(2)不同的进化性机制是否对单药和多药耐药性起作用，以及(3)破坏这些基因是否可以维持或恢复抗生素敏感性知之甚少。为了解决这些问题，我们将使用一种称为“比较实验进化”的多学科方法，这是一种强大的技术，能够研究基因组含量的物种水平差异如何影响细菌进化耐药性的能力。该方法将高通量实验进化和细菌表型与全基因组测序和比较基因组学相结合。我们将在单一和多种抗生素的环境下，从8种假单胞菌中进化出近6万个独立的种群。我们将把基因组内容的差异与每个物种获得的具有高水平抗性的突变的差异联系起来。然后，我们将使用现代基因组编辑技术，看看破坏可进化性基因是否可以限制耐药性，或者恢复已经具有耐药性的生物体的敏感性。这些大规模平行实验将揭示耐药性进化的基因组基础，同时也揭示高水平耐药性与染色体突变之间的联系。这个项目将提高我们对这些经济上重要的细菌的耐药性进化的认识。它还将提供一个框架，从中我们可以识别预测耐药性进化风险的遗传标记，从而更好地有针对性地使用抗菌素。最后，它将为测试抗进化性方法提供一个框架，以防止抗性和恢复敏感性。

The growing prevalence of antibiotic resistance is a major crisis for both public health and agriculture. Bacteria can dramatically vary in their ability to evolve resistance, but our ability to predict which ones will go on to evolve high-level resistance is currently limited. Understanding what contributes to the evolutionary potential for resistance will enable us to develop new interventions for suppressing antimicrobial resistance. It is therefore important to understand the genomic mechanisms that contribute to the 'evolvability' of resistance.We will investigate how genome diversity contributes to the ability to evolve resistance. In contrast to other work that focuses on a single species of bacteria, we will investigate how between-species genome diversity contributes to the ability to evolve resistance. We will focus on Pseudomonas bacteria, an incredibly diverse genus that includes environmental, commensal and pathogenic organisms. Pseudomonas imposes a global economic burden that exceeds £150 billion GBP annually across health and agricultural sectors. Antibiotic resistance in Pseudomonas is increasing rapidly, and understanding what allows resistance to evolve to important anti-pseudomonal antibiotics is key to maintaining the ability to manage Pseudomonas. We will therefore determine how genome-level variation contributes to the evolvability of resistance to anti-pseudomonal antibiotics, including one recently come to market specifically designed to target pseudomonad physiology (cefiderocol).Evidence from Pseudomonas suggests that even seemingly minor differences in genome content can have extensive consequences for the potential to evolve resistance. Previous work has shown that a single 'evolvability gene' can influence whether pseudomonads can evolve high-level resistance to the antibiotic ceftazidime. However, we currently do not know the extent to which such mechanisms generally operate. Specifically, little is known about (i) how resistance evolvability varies across diverse antibiotic classes, (ii) whether different evolvability mechanisms operate for single- and multi-drug resistance, and (iii) whether disrupting such genes can maintain or restore antibiotic sensitivity.To address these questions, we will use a multi-disciplinary approach called 'comparative experimental evolution', a powerful technique able to investigate how species-level differences in genome content affect the ability for bacteria to evolve resistance. This approach combines high-throughput experimental evolution and bacterial phenotyping with whole genome sequencing and comparative genomics. We will evolve nearly 60,000 independent populations from eight Pseudomonas species under single- and multiple-antibiotic environments. We will connect differences in genome content to differences in mutations acquired by each species that confer high-level resistance. We will then use modern genome editing techniques to see if disrupting evolvability genes can constrain resistance, or restore sensitivity in already-resistant organisms. These massively parallel experiments will reveal the genomic basis for resistance evolvability, while also revealing the connection between high-level resistance and chromosomal mutations. This project will advance our knowledge of what potentiates resistance evolution in these economically-important bacteria. It will also provide a framework from which we can identify genetic markers for predicting the risk of resistance evolution, allowing better targeted use of antimicrobials. Finally, it will provide a framework for testing anti-evolvability approaches to preventing resistance and restoring sensitivity.