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.

Keywords: Antibiotic; Fluorescence-activated cell sorting (FACS); Persister; Phenotypic heterogeneity; Redox sensor green (RSG); Viable but non-culturable cell (VBNC).

Figure 1. Biphasic killing of E. coli treated with antibiotics

Survival fraction of exponential phase E. coli treated with 200µg/mL ampicillin (AMP) or 5µg/mL ofloxacin (OFL) as measured by CFU. Initial phase of killing (I: dark gray) corresponds to death of normal cells, whereas the second phase of killing (II: light gray) represents colonies derived from persisters.

Figure 2. FACS method to study persister metabolism

(A) Exponential phase cells (E. coli MG1655::ΔcyoA) were stained with RSG. RSG produces a stable green fluorescent signal when reduced by bacterial reductases. Staining was diminished when cells were pre-treated with CCCP, which depletes proton motive force. (B) RSG stained cells were sorted from the indicated regions (gates) in order to quantify the persister distribution within the quantiles. Gates A, B, C and D compromise 10, 40, 40, and 10%, respectively, of the entire population. (C–D) Persister frequencies were quantified after 5h antibiotic treatment of FACS sorted cells from regions A, B, C, and D. The frequency is the ratio of persisters to initial number of FACS sorted cells. (E–F) Persister frequencies in control samples were similarly quantified after 5h antibiotic treatment. “Entire population (sorted)” corresponds to samples that were sorted without gating, “Entire population (unsorted)” corresponds to samples that did not enter the sorter, and “Recovery (calculated)” is the frequency of persisters one would expect from the total population, as calculated from the persister frequencies measured from the segregated quantiles (A, B, C, D). We note that these three quantities should be indistinguishable from one another. Genetic deletion for MG1655::ΔcyoA strain was transduced from the Keio collection using the standard P1 phage method (33) and the mutation was confirmed with PCR.

Figure 3. SSC-A vs. FSC-A dot-plot

FSC and SSC PMT voltage settings are adjusted so that exponential phase E. coli cells (unstained, non-FP expressing) are on SSC-A and FSC-A scales. Each dot represents a cell.