Abstract
Resistance to clinically useful therapeutic antibiotics is an ever-increasing phenomenon seen in a range of bacterial species including those pathogenic to man. There are diverse mechanisms which contribute to inherent and acquired resistance to antibiotics. Gram-negative bacteria are commonly intrinsically more resistant to many drugs as a result of their cell structure and the activity of multidrug efflux pumps. Measurement of the accumulation of antibiotics and the contribution of active efflux has proved important in understanding the mechanisms of resistance to many antibiotics and how bacteria can become multidrug-resistant. Multidrug efflux pumps often have broad substrate ranges allowing detection of their activity by measurement of the accumulation of antibiotic substrates or a range of fluorescent substrates, which can be easily used as markers of efflux activity. This chapter describes methods for the detection of efflux pump activity on Gram-negative bacteria.
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References
Nikaido H (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67:593–656
Zgurskaya HI, Nikaido H (2000) Multidrug resistance mechanisms: drug efflux across two membranes. Mol Microbiol 37:219–225
Kaatz GW (2005) Bacterial efflux pump inhibition. Curr Opin Investig Drugs 6:191–198
Piddock LJ (2006) Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin Microbiol Rev 19:382–402
Tseng TT, Gratwick KS, Kollman J, Park D, Nies DH, Goffeau A, Saier MH Jr (1999) The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. J Mol Microbiol Biotechnol 1:107–125
Mortimer PG, Piddock LJ (1991) A comparison of methods used for measuring the accumulation of quinolones by Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. J Antimicrob Chemother 28:639–653
Notarianni LJ, Jones RW (1988) Method for the determination of ofloxacin, a quinolone carboxylic acid antimicrobial, by high-performance liquid chromatography. J Chromatogr 431:461–464
Neyfakh AA, Bidnenko VE, Chen LB (1991) Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system. Proc Natl Acad Sci U S A 88:4781–4785
Babayan A, Nikaido H (2004) In Pseudomonas aeruginosa ethidium bromide does not induce its own degradation or the assembly of pumps involved in its efflux. Biochem Biophys Res Comm 324:1065–1068
Acknowledgments
The authors would like to thank Professors Martin Woodward and Laura Piddock for useful input during development of this method and Defra for financial support (project number OD2011).
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Webber, M.A., Coldham, N.G. (2010). Measuring the Activity of Active Efflux in Gram-Negative Bacteria. In: Gillespie, S., McHugh, T. (eds) Antibiotic Resistance Protocols. Methods in Molecular Biology, vol 642. Humana Press. https://doi.org/10.1007/978-1-60327-279-7_13
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DOI: https://doi.org/10.1007/978-1-60327-279-7_13
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