A robust method for generating, quantifying, and testing large numbers of escherichia coli persisters.

Bacteria can exhibit phenotypes that render them tolerant against antibiotics. However, often only a few cells of a bacterial population show the so-called persister phenotype, which makes it difficult to study this health-threatening phenotype. We recently found that certain abrupt nutrient shifts generate Escherichia coli populations that consist almost entirely of antibiotic-tolerant cells. These nearly homogeneous persister cell populations enable assessment with population-averaging experimental methods, such as high-throughput methods. In this chapter, we provide a detailed protocol for generating a large fraction of tolerant cells using the nutrient-switch approach. Furthermore, we describe how to determine the fraction of cells that enter the tolerant state upon a sudden nutrient shift and we provide a new way to assess antibiotic tolerance using flow cytometry. We envision that these methods will facilitate research into the important and exciting phenotype of bacterial persister cells

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis.

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters