High-throughput flow cytometry screening of multidrug efflux systems

Mark K. Haynes, Matthew Garcia, Ryan Peters, Anna Waller, Pietro Tedesco, Oleg Ursu, Cristian G. Bologa, Radleigh G. Santos, Clemencia Pinilla, Terry H. Wu, Julie A. Lovchik, Tudor I. Oprea, Larry A. Sklar, George P. Tegos

Research output: Chapter in Book/Report/Conference proceedingChapter

9 Scopus citations


The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages26
StatePublished - 2018
Externally publishedYes

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745

Bibliographical note

Publisher Copyright:
© 2018, Springer Science+Business Media LLC.


  • Antibiotic enhancement
  • Biological Warfare bacterial agents
  • Burkholderia sp
  • Flow cytometry
  • Francisella sp
  • RND efflux transporters


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