Flow cytometric analysis of efflux by dye accumulation

Research output: Contribution to journalArticle

Authors

  • Emily E. Whittle
  • Simon W. Legood
  • Ilyas Alav
  • Punyawee Dulyayangkul

Abstract

Gram-negative infections are increasingly difficult to treat because of their impermeable outer membranes (OM) and efflux pumps which maintain a low intracellular accumulation of antibiotics within cells. Historically, measurement of accumulation of drugs or dyes within Gram-negative cells has concentrated on analyzing whole bacterial populations. Here, we have developed a method to measure the intracellular accumulation of ethidium bromide, a fluorescent DNA intercalating dye, in single cells using flow cytometry. Bacterial cells were stained with SYTOTM 84 to easily separate cells from background cell debris. Ethidium bromide fluorescence was then measured within the SYTOTM 84 positive population to measure accumulation. In S. Typhimurium SL1344, ethidium bromide accumulation was low, however, in a number of efflux mutants, accumulation of ethidium bromide increased more than twofold, comparable to previous whole population analysis of accumulation. We demonstrate simultaneous measurement of ethidium bromide accumulation and GFP allowing quantification of gene expression or other facets of phenotype in single cells. In addition, we show here that this assay can be adapted for use with efflux inhibitors, with both Gram-negative and Gram-positive bacteria, and with other fluorescent substrates with different fluorescence spectra.

Bibliographic note

Copyright © 2019 Whittle, Legood, Alav, Dulyayangkul, Overton and Blair.

Details

Original languageEnglish
Article number2319
Number of pages10
JournalFrontiers in Microbiology
Volume10
Publication statusPublished - 4 Oct 2019

Keywords

  • flow cytometry, accumulation, efflux, ethidium bromide, gram-negative