Elucidation of the genetic determinants of antibiotic tolerance

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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.