title: Bacterial efflux pump inhibitors.
abstract: 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 over 24 h.
authors.affiliation: Pfizer Global R & D, MS, Groton, CT, USA.
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Kamicker
authors.forename: Michael T
authors.initials: MT
authors.lastname: Sweeney
authors.forename: Frank
authors.initials: F
authors.lastname: Kaczmarek
authors.forename: Fadia
authors.initials: F
authors.lastname: Dib-Hajj
authors.forename: Wenchi
authors.initials: W
authors.lastname: Shang
authors.forename: Kim
authors.initials: K
authors.lastname: Crimin
authors.forename: Joan
authors.initials: J
authors.lastname: Duignan
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Gootz
mesh_terms: D000900:Anti-Bacterial Agents / Q000032:analysis* / Q000302:isolation & purification / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors; D001426:Bacterial Proteins / Q000032:analysis / Q000037:antagonists & inhibitors; D001693:Biological Transport, Active / Q000187:drug effects*; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects; D004996:Ethidium / Q000378:metabolism; D006193:Haemophilus influenzae / Q000187:drug effects; D006801:Humans; D008826:Microbial Sensitivity Tests; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors*
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D004996:Ethidium; D027425:ATP-Binding Cassette, Sub-Family C Proteins