title: Analyzing Persister Physiology with Fluorescence-Activated Cell Sorting.
abstract: Bacterial persisters are phenotypic variants that exhibit an impressive ability to tolerate antibiotics. Persisters are hypothesized to cause relapse infections, and therefore, understanding their physiology may lead to novel therapeutics to treat recalcitrant infections. However, persisters have yet to be isolated due to their low abundance, transient nature, and similarity to the more highly abundant viable but non-culturable cells (VBNCs), resulting in limited knowledge of their phenotypic state. This technical hurdle has been addressed through the use of fluorescence-activated cell sorting (FACS) and quantification of persister levels in the resulting sorted fractions. These assays provide persister phenotype distributions, which can be compared to the phenotype distributions of the entire population, and can also be used to examine persister heterogeneity. Here, we describe two detailed protocols for analysis of persister physiology with FACS. One protocol assays the metabolic state of persisters using a fluorescent metabolic stain, whereas the other assays the growth state of persisters with use of a fluorescent protein.
authors.affiliation: Department of Chemical and Biological Engineering, Princeton University, 205 Hoyt Laboratory, Princeton, NJ, 08544, USA.
authors.forename: Mehmet A
authors.initials: MA
authors.lastname: Orman
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA.|Rutgers Robert Wood Johnson Medical School, Piscataway, NJ, USA.
authors.forename: Theresa C
authors.initials: TC
authors.lastname: Henry
authors.affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
authors.forename: Christina J
authors.initials: CJ
authors.lastname: DeCoste
authors.affiliation: Department of Chemical and Biological Engineering, Princeton University, 205 Hoyt Laboratory, Princeton, NJ, 08544, USA. mbrynild@princeton.edu.|Department of Molecular Biology, Princeton University, Princeton, NJ, USA. mbrynild@princeton.edu.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Brynildsen
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents
keywords: Antibiotic; Fluorescence-activated cell sorting (FACS); Persister; Phenotypic heterogeneity; Redox sensor green (RSG); Viable but non-culturable cell (VBNC)