rifampin and tetraphenylphosphonium

AcrAB-TolC is the major constitutively expressed efflux pump system that provides resistance to a variety of antimicrobial agents and dyes in Escherichia coli. However, no systematically optimized real-time dye efflux assay has been published for the measurement of its activity and for detection of possible competition between substrates. Here, we report on the development of such an assay using a lipophilic dye, Nile Red. Energy-depleted cells were loaded with the dye in the presence of low (10 microM or less) concentrations of the proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP). The CCCP was then removed, and efflux was triggered by energization with glucose. Various known efflux pump inhibitors and antimicrobials were checked for the ability to slow down Nile Red efflux, presumably through competition. Besides the known inhibitors Phe-Arg-beta-naphthylamide and 1-naphthyl-methylpiperazine, several tetracyclic compounds (doxorubicin, minocycline, chlortetracycline, doxycycline, and tetracycline) and tetraphenylphosphonium chloride were found to interfere with dye efflux. This inhibition could not be explained by the depletion of proton motive force. None of the other tested antimicrobials, including macrolides, fluoroquinolones, and beta-lactams, had any impact on Nile Red efflux, even at concentrations of up to 1 mM.

Topics: Anti-Bacterial Agents; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Chlortetracycline; Dipeptides; Doxorubicin; Doxycycline; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Proteins; Minocycline; Onium Compounds; Organophosphorus Compounds; Tetracycline

A multidrug efflux pump designated LmrS (lincomycin resistance protein of Staphylococcus aureus), belonging to the major facilitator superfamily (MFS) of transporters, was cloned, and the role of LmrS in antimicrobial efflux was evaluated. The highest relative increase in MIC, 16-fold, was observed for linezolid and tetraphenylphosphonium chloride (TPCL), followed by an 8-fold increase for sodium dodecyl sulfate (SDS), trimethoprim, and chloramphenicol. LmrS has 14 predicted membrane-spanning domains and is homologous to putative lincomycin resistance proteins of Bacillus spp., Lactobacillus spp., and Listeria spp.

Topics: Amino Acid Sequence; Bacterial Proteins; Chloramphenicol; Drug Resistance, Multiple, Bacterial; Ethidium; Membrane Transport Proteins; Microbial Sensitivity Tests; Molecular Sequence Data; Onium Compounds; Organophosphorus Compounds; Phylogeny; Sequence Homology, Amino Acid; Sodium Dodecyl Sulfate; Staphylococcus aureus; Trimethoprim; Vancomycin