Determining the architecture of antibiotic resistance evolvability

抗生素耐药性的日益普遍是公共卫生和农业的一个重大危机。细菌进化耐药性的能力可能会有很大的差异，但我们预测哪些细菌会进化出高水平耐药性的能力目前还很有限。了解是什么导致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生素耐药性。因此，了解基因组机制有助于抗性的“可进化性”是很重要的。我们将研究基因组多样性如何有助于抗性的进化能力。与其他专注于单一细菌物种的工作相反，我们将研究物种间基因组多样性如何有助于进化抗性的能力。我们将专注于假单胞菌，一个令人难以置信的多样化的属，包括环境，微生物和病原生物。假单胞菌每年在卫生和农业部门造成超过1500亿英镑的全球经济负担。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化如何有助于抗假单胞菌抗生素的耐药性的进化，包括最近上市的专门针对假单胞菌生理学（cefiderocol）的证据表明，即使是看似微小的差异，基因组内容可以有广泛的后果的潜力，发展耐药性。以前的研究表明，一个单一的“进化性基因”可以影响假单胞菌是否可以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，我们对以下问题知之甚少：（i）不同抗生素类别之间的耐药性进化性如何变化，（ii）不同的进化机制是否对单药和多药耐药性起作用，以及（iii）破坏这些基因是否可以维持或恢复抗生素敏感性。为了解决这些问题，我们将使用称为“比较实验进化”的多学科方法，这是一项强大的技术，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。这种方法将高通量实验进化和细菌表型分析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环境下从8种假单胞菌中进化出近60，000个独立种群。我们将把基因组内容的差异与每个物种获得的赋予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，看看破坏进化性基因是否可以限制抗性，或者恢复已经具有抗性的生物体的敏感性。这些大规模平行实验将揭示抗性可进化性的基因组基础，同时也揭示高水平抗性与染色体突变之间的联系。该项目将推进我们对这些经济上重要的细菌中增强抗性进化的知识。它还将提供一个框架，我们可以从中识别遗传标记来预测耐药性演变的风险，从而更好地有针对性地使用抗菌药物。最后，它将提供一个框架，用于测试防止抗性和恢复敏感性的反进化方法。

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.