title: Evaluation of efflux activity of bacteria by a semi-automated fluorometric system.
abstract: A semi-automated method that uses the common efflux pump (EP) substrate ethidium bromide (EB) is described for the assessment of EP systems of bacteria. The method employs the Rotor-Gene(TM) 3000 thermocycler (Corbett Research) for the real-time assessment of accumulation and efflux of EB in Phosphate-Buffered Solution (PBS) under varying physiological conditions, such as temperature, pH, presence and absence of the energy source, and presence of efflux pumps inhibitors (EPIs). The method is sufficiently sensitive to characterize intrinsic EP systems of reference strains, a prime necessity if there is a need for assessment of EP-mediated multi-drug resistance (MDR). The method has been successfully applied by us to characterize intrinsic and over-expressed EP systems of Escherichia coli, Salmonella Enteritidis, Enterobacter aerogenes, Enterococcus faecalis and Enterococcus faecium, Staphylococcus aureus, and Mycobacterium smegmatis and Mycobacterium avium, suggesting that if the organism can be maintained in PBS, the system described may suffice for the evaluation and assessment of its EP system.
authors.affiliation: Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisboa, Portugal.
authors.forename: Miguel
authors.initials: M
authors.lastname: Viveiros
authors.forename: Liliana
authors.initials: L
authors.lastname: Rodrigues
authors.forename: Marta
authors.initials: M
authors.lastname: Martins
authors.forename: Isabel
authors.initials: I
authors.lastname: Couto
authors.forename: Gabriella
authors.initials: G
authors.lastname: Spengler
authors.forename: Ana
authors.initials: A
authors.lastname: Martins
authors.forename: Leonard
authors.initials: L
authors.lastname: Amaral
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D024901:Drug Resistance, Multiple, Bacterial / Q000502:physiology; D004996:Ethidium / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D026901:Membrane Transport Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D004996:Ethidium