Elucidation of the genetic determinants of antibiotic tolerance

阐明抗生素耐受性的遗传决定因素

• 批准号: RGPIN-2022-04577
• 负责人: Stokes, Jonathan
• 金额: $ 2.26万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744185
• 关键词: Elucidation; genetic; determinants; antibiotic; tolerance

The genetic determinants of antibiotic resistance are well-studied. Indeed, a wide range of resistance determinants have been discovered that collectively confer resistance to all known antibiotics. In addition to resistance, antibiotic tolerance is a distinct - and poorly understood - mechanism through which bacteria can survive antibiotic exposure. Antibiotic tolerant cells are those that do not harbour resistance genes yet are able to survive exposure to lethal concentrations of antibiotics. This phenotype is often due to some form of metabolic repression of a subpopulation of an actively growing culture, or the metabolic repression of an entire culture in nutrient-deplete conditions, such as existence within biofilms.  While some progress has been made towards identifying the biological processes that govern antibiotic tolerance in specific conditions, a systematic exploration into the totality of genetic mechanisms of tolerance across a wide range of antibiotic classes has yet to be conducted. We propose to leverage a combination of laboratory evolution, genetic screening, and computational approaches to systematically uncover the genetic determinants of antibiotic tolerance. Initially, we will explore the genetic determinants of antibiotic tolerance in the model organism Escherichia coli. Aim 1. Acquisition of antibiotic tolerant mutants of E. coli We will leverage a combination of laboratory evolution and genetic mutant library screening to acquire antibiotic tolerant populations of E. coli against molecules from four antibiotic classes: ß-lactams, aminoglycosides, fluoroquinolones, and polymyxins. Aim 2. Discovery and validation of tolerance mutations from non-laboratory isolates After collecting a diverse set of mutant strains, we will computationally curate environmental and clinical E. coli genomes with the goal of leveraging our mutant data from Aim 1 to identify all potential tolerance-conferring mutations. Aim 3. Elucidating the relationships between tolerance and resistance Once we have discovered and validated a comprehensive collection of antibiotic tolerance mutations, we will identify all known antibiotic resistance genes within each strain - both laboratory-acquired strains and computationally curated strains. Next, on a strain-by-strain basis, we will quantify the co-existence of every tolerance mutation with every resistance gene to determine whether specific resistance genes are enriched in specific tolerant genetic backgrounds. Our long-term research will expand these efforts to a wide spectrum of bacterial species and antibiotics, with the goal of developing a strong understanding of the genetic basis of antibiotic tolerance, as well as the relationship between tolerance and resistance. This will provide useful insight into the breadth of possible bacterial survival strategies, which will lead to more rational use of antibiotics in agricultural and clinical settings, and guide future antibiotic development efforts.

抗生素耐药性的遗传决定因素已得到充分研究。事实上，已经发现了广泛的耐药决定簇，它们共同赋予对所有已知抗生素的耐药性。除了耐药性之外，抗生素耐受性是一种独特的机制，但人们对其知之甚少，通过这种机制，细菌可以在抗生素暴露中存活下来。抗生素耐受细胞是那些不携带抗性基因但能够在暴露于致死浓度的抗生素中存活的细胞。这种表型通常是由于活跃生长的培养物的亚群的某种形式的代谢抑制，或者在营养耗尽的条件下整个培养物的代谢抑制，例如存在于生物膜内。对广泛的抗生素类别的耐受性的遗传机制的整体进行系统的探索还有待进行。我们建议利用实验室进化、遗传筛选和计算方法的组合来系统地揭示抗生素耐受性的遗传决定因素。首先，我们将探讨模式生物大肠杆菌抗生素耐受性的遗传决定因素。目标1。获得了耐抗生素突变株。我们将利用实验室进化和遗传突变库筛选相结合的方法获得耐抗生素的大肠杆菌群体。大肠杆菌对来自四种抗生素类的分子：β-内酰胺类、氨基糖苷类、氟喹诺酮类和多粘菌素类。目标2.从非实验室分离株中发现和验证耐受性突变在收集了一组不同的突变株后，我们将通过计算筛选环境和临床E。大肠杆菌基因组，目的是利用我们的突变体数据从目标1，以确定所有潜在的耐受性赋予突变。目标3.阐明耐药性和耐药性之间的关系一旦我们发现并验证了抗生素耐药性突变的全面集合，我们将确定每个菌株中所有已知的抗生素耐药性基因-实验室获得的菌株和计算筛选的菌株。接下来，在菌株的基础上，我们将量化每个耐受性突变与每个抗性基因的共存，以确定特定的抗性基因是否在特定的耐受性遗传背景中富集。我们的长期研究将把这些努力扩展到广泛的细菌物种和抗生素，目标是深入了解抗生素耐受性的遗传基础，以及耐受性和耐药性之间的关系。这将为可能的细菌生存策略的广度提供有用的见解，这将导致在农业和临床环境中更合理地使用抗生素，并指导未来的抗生素开发工作。