High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems
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.
Key wordsFlow cytometry RND efflux transporters Antibiotic enhancement Biological Warfare bacterial agents Burkholderia sp. Francisella sp.
This work was supported by The Department of Threat and Reduction Agency (HDTRA1-13-C-0005 to G.P.T. and L.A.S.) and The UNM Clinical and Translational Science Center (UL1TR001449 to L.A.S.). The authors thank the Italian Research Council for a Fellowship to Pietro Tedesco.
- 7.Nakashima R, Sakurai K, Yamasaki S et al (2011) Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket. Nature 27:565–569Google Scholar
- 11.Mima T, Schweizer HP (2010) The BpeAB-OprB efflux pump of Burkholderia pseudomallei 1026b does not play a role in quorum sensing, virulence factor production, or extrusion of aminoglycosides but is a broad-spectrum drug efflux system. Antimicrob Agents Chemother 54:3113–3120CrossRefPubMedPubMedCentralGoogle Scholar
- 24.Hoel DG (1987) Statistical aspects of chemical mixtures. In: Vouk VB, Butler GC, Upton AC, Parke DV, Asher SC (eds) Methods for assessing the effects of mixtures of chemicals. Wiley, New York, pp 369–377Google Scholar
- 35.Clinical Laboratory Standards Institute (2007) M7-A7. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard, 7th edn. CLSI, Wayne, PAGoogle Scholar
- 39.Coban AY, Tanriverdi-Cayci Y, Erturan Z et al (2009) Effects of efflux pump inhibitors phenyl-arginine-beta-naphthylamide and 1-(1-naphthylmethyl)-piperazine on the antimicrobial susceptibility of Pseudomonas aeruginosa isolates from cystic fibrosis patients. J Chemother 21:592–594CrossRefPubMedGoogle Scholar
- 40.Schumacher A, Steinke P, Bohnert JA et al (2006) Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Enterobacteriaceae other than Escherichia coli. J Antimicrob Chemother 57:344–348CrossRefPubMedGoogle Scholar