Elucidation of the genetic determinants of antibiotic tolerance

抗生素耐药性的遗传决定因素已经得到了很好的研究。事实上，已经发现了广泛的耐药性决定因素，它们共同赋予了对所有已知抗生素的耐药性。除了耐药性，抗生素耐受是细菌在抗生素暴露下生存的一种独特的--也是鲜为人知的--机制。抗生素耐受细胞是指那些没有耐药基因但能够在致命浓度的抗生素下存活的细胞。这种表型通常是由于活跃生长的培养物的一个亚群的某种形式的代谢抑制，或者是整个培养物在营养耗竭的条件下的代谢抑制，例如在生物膜内的存在。尽管在确定在特定条件下支配抗生素耐受性的生物过程方面已经取得了一些进展，但对广泛的抗生素类别的耐受性的遗传机制的整体的系统探索还没有进行。我们建议利用实验室进化、基因筛选和计算方法的组合来系统地揭示抗生素耐药性的遗传决定因素。首先，我们将探索模式生物大肠杆菌中抗生素耐受性的遗传决定因素。目的1.大肠杆菌耐药突变株的获得我们将利用实验室进化和基因突变体库筛选相结合的方法，获得对来自四种抗生素类别的分子：β-内酰胺类、氨基糖苷类、氟喹诺酮类和多粘菌素的耐药菌群。目的2.非实验室分离株耐受性突变的发现和验证在收集了一组不同的突变菌株后，我们将对环境和临床大肠杆菌基因组进行计算，目的是利用我们来自Aim 1的突变数据来识别所有潜在的耐受性突变。目的3.阐明耐受性和耐药性之间的关系一旦我们发现并验证了抗生素耐受性突变的全面集合，我们将识别每个菌株中所有已知的抗生素耐药性基因--包括实验室获得的菌株和计算筛选的菌株。接下来，我们将在逐个菌株的基础上，量化每个耐药突变与每个抗性基因的共存情况，以确定特定的抗性基因是否在特定的耐药遗传背景中得到丰富。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，目标是对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有一个强有力的理解。这将为可能的细菌生存策略的广度提供有用的洞察，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。

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.