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-GeneTM 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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Poole K (2007) Efflux pumps as antimicrobial resistance mechanisms. Ann Med 39:162–176
Levy SB, McMurry L (1978) Plasmid-determined tetracycline resistance involves new transport systems for tetracycline. Nature 276:90–92
McMurry L, Petrucci RE, Levy SB (1980) Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. Proc Natl Acad Sci USA 77:3974–3977
Ball PR, Shales SW, Chopra I (1980) Plasmid-mediated tetracycline resistance in Escherichia coli involves increased efflux of the antibiotic. Biochem Biophys Res Commun 93:74–81
Nikaido H (1988) Bacterial resistance to antibiotics as a function of outer membrane permeability. J Antimicrob Chemother 22:17–22
Gootz TD (2006) The forgotten Gram-negative bacilli: what genetic determinants are telling us about the spread of antibiotic resistance. Biochem Pharmacol 71:1073–1084
Poole K (2003) Overcoming multidrug resistance in gram-negative bacteria. Curr Opin Investig Drugs 4:128–139
Giuliodori AM, Gualerzi CO, Soto S, Vila J, Tavío MM (2007) Review on bacterial stress topics. Ann NY Acad Sci 1113:95–104
Martins M, Dastidar SG, Fanning S, Kristiansen JE, Molnar J, Pagès JM et al (2008) Potential role of non-antibiotics (helper compounds) in the treatment of multidrug-resistant Gram-negative infections: mechanisms for their direct and indirect activities. Int J Antimicrob Agents 31:198–208
Piddock LJ (2006) Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin Microbiol Rev 19:382–402
Lomovskaya O, Bostian KA (2006) Practical applications and feasibility of efflux pump inhibitors in the clinic – a vision for applied use. Biochem Pharmacol 71:910–918
Viveiros M, Dupont M, Rodrigues L, Couto I, Davin-Regli A, Martins M et al (2007) Antibiotic stress, genetic response and altered permeability of E. coli. PLoS One 2:e365
Baquero F (2001) Low-level antibacterial resistance: a gateway to clinical resistance. Drug Resist Updat 4:93–105
Martinez JL, Baquero F, Andersson DI (2007) Predicting antibiotic resistance. Nat Rev Microbiol 5:958–965
Lomovskaya O, Zgurskaya HI, Totrov M, Watkins WJ (2007) Waltzing transporters and “the dance macabre” between humans and bacteria. Nat Rev Drug Discov 6:56–65
Piddock LJ (2006) Multidrug-resistance efflux pumps – not just for resistance. Nat Rev Microbiol 4:629–636
Viveiros M, Martins A, Paixão L, Rodrigues L, Martins M, Couto I et al (2008) Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method. Int J Antimicrob Agents 31:458–462
Rodrigues L, Wagner D, Viveiros M, Sampaio D, Couto I, Vavra M et al (2008) Thioridazine and chlorpromazine inhibition of ethidium bromide efflux in Mycobacterium avium and Mycobacterium smegmatis. J Antimicrob Chemother 61:1076–1082
Couto I, Costa SS, Viveiros M, Martins M, Amaral L (2008) Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide. J Antimicrob Chemother 62:04–513
Greulich KO (2004) Single molecule techniques for biomedicine and pharmacology. Curr Pharm Biotechnol 5:243–259
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
Hendrikse NH (2000) Monitoring interactions at ATP-dependent drug efflux pumps. Curr Pharm Des 6:1653–1668
Schumacher A, Trittler R, Bohnert JA, Kummerer K, Pagès JM, Kern WV (2007) Intracellular accumulation of linezolid in Escherichia coli, Citrobacter freundii and Enterobacter aerogenes: role of enhanced efflux pump activity and inactivation. J Antimicrob Chemother 59:1261–1264
Shapiro HM (2008) Flow cytometry of bacterial membrane potential and permeability. Methods Mol Med 142:175–186
Aszalos A (2007) Drug-drug interactions affected by the transporter protein, P-glycoprotein (ABCB1, MDR1) I. Preclinical aspects. Drug Discov Today 12:833–837
Kyriacou SV, Nowak ME, Brownlow WJ, Xu XHN (2002) Single live cell imaging for real-time monitoring of resistance mechanism in Pseudomonas aeruginosa. J Biomed Opt 7:576–586
Ryan BM, Dougherty TJ, Beaulieu D, Chuang J, Dougherty BA, Barrett JF (2001) Efflux in bacteria: what do we really know about it? Expert Opin investig Drugs 10:1409–1422
Joux F, Lebaron P (2000) Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes Infect 2:1523–1535
Jernaes MW, Steen HB (1994) Staining of Escherichia coli for flow cytometry: influx and efflux of ethidium bromide. Cytometry 17:302–309
Duffy L, Dykes GA (2006) Growth temperature of four Campylobacter jejuni strains influences their subsequent survival in food and water. Lett Appl Microbiol 43:596–601
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, USA
Acknowledgments
This work was supported by grants EU-FSE/FEDER-POCI /SAU-MMO/59370/2004 and EU-FSE/FEDER-PTDC/BIA-MIC/71280/2006 provided by the Fundação para a Ciência e a Tecnologia (FCT) of Portugal. M. Martins, A. Martins, L. Rodrigues, and G. Spengler were supported by grants SFRH/BD/14319/2003, SFRH/BD/19445/2004, SFRH/BD/24931/2005, and SFRH/BPD/34578/2007 from the FCT of Portugal, respectively. The authors wish to thank Séamus Fanning, Winfried V. Kern, and Jean-Marie Pagès for the MDR strains they have provided for evaluation of efflux activity, as well as for the many discussions that stimulated aspects of this study.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Viveiros, M. et al. (2010). Evaluation of Efflux Activity of Bacteria by a Semi-automated Fluorometric System. 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_12
Download citation
DOI: https://doi.org/10.1007/978-1-60327-279-7_12
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-278-0
Online ISBN: 978-1-60327-279-7
eBook Packages: Springer Protocols