Summary
Infections caused by multidrug-resistant Gram-negative pathogens play a major role in the morbidity and mortality of hospitalized patients. The rise of resistance to current antibiotic therapies has made the discovery of new agents urgent. One of the major antibiotic resistance mechanisms utilized by more than 15 species of Gram-negative bacterial cells is the Resistance Nodulation Division (RND) efflux pump, which eliminates several classes of antibiotics such as penicillins and cephalosporin macrolides aminoglycosides, fluoroquinolonesx and tetracyclines. Here we describe a multistep process to identify compounds that inhibit the RND-type efflux pumps. This involves measuring the inhibition of accumulation of ethidium bromide in E. coli or Haemophilus influenzae cells and confirming that the inhibition is specific for the efflux pumps by using genetic constructs and biochemical methods to measure nonspecific inhibition due to e.g. intrinsic antibacterial activity or membrane disruption. In whole bacterial cells synergism antagonism or indifference of the combination of an antibiotic with the putative inhibitor is determined and this is then confirmed by quantitating viable bacterial cells in liquid culture over24h.
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Acknowledgments
We acknowledge the work of numerous former colleagues at Pharmacia who started this research program: D. E. Decker M. P. Sheets S. E. Buxser M. R. Barbachyn R.Gadwood G.Zurenko J. Bourdage G. F. Hess J. K. Gibson A. G. Morgan J. E. Mott etal. We also thank Keith Poole and colleagues at Queens University in Kingston Ontario for E. coli strain constructs.
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Kamicker, B.J. et al. (2008). Bacterial Efflux Pump Inhibitors. In: Champney, W.S. (eds) New Antibiotic Targets. Methods In Molecular Medicine™, vol 142. Humana Press. https://doi.org/10.1007/978-1-59745-246-5_15
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DOI: https://doi.org/10.1007/978-1-59745-246-5_15
Publisher Name: Humana Press
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