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Evaluation of Efflux Activity of Bacteria by a Semi-automated Fluorometric System

  • Miguel Viveiros
  • Liliana Rodrigues
  • Marta Martins
  • Isabel Couto
  • Gabriella Spengler
  • Ana Martins
  • Leonard AmaralEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 642)

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.

Key words

Efflux pumps Efflux pump inhibitors Permeability Ethidium bromide Semi-automated fluorometric method 

Notes

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.

References

  1. 1.
    Poole K (2007) Efflux pumps as antimicrobial resistance mechanisms. Ann Med 39:162–176CrossRefPubMedGoogle Scholar
  2. 2.
    Levy SB, McMurry L (1978) Plasmid-determined tetracycline resistance involves new transport systems for tetracycline. Nature 276:90–92CrossRefPubMedGoogle Scholar
  3. 3.
    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 77:3974–3977CrossRefPubMedGoogle Scholar
  4. 4.
    Ball PR, Shales SW, Chopra I (1980) Plasmid-mediated tetracycline resistance in Escherichia coli involves increased efflux of the antibiotic. Biochem Biophys Res Commun 93:74–81CrossRefPubMedGoogle Scholar
  5. 5.
    Nikaido H (1988) Bacterial resistance to antibiotics as a function of outer membrane permeability. J Antimicrob Chemother 22:17–22PubMedGoogle Scholar
  6. 6.
    Gootz TD (2006) The forgotten Gram-negative bacilli: what genetic determinants are telling us about the spread of antibiotic resistance. Biochem Pharmacol 71:1073–1084CrossRefPubMedGoogle Scholar
  7. 7.
    Poole K (2003) Overcoming multidrug resistance in gram-negative bacteria. Curr Opin Investig Drugs 4:128–139PubMedGoogle Scholar
  8. 8.
    Giuliodori AM, Gualerzi CO, Soto S, Vila J, Tavío MM (2007) Review on bacterial stress topics. Ann NY Acad Sci 1113:95–104CrossRefPubMedGoogle Scholar
  9. 9.
    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 31:198–208CrossRefPubMedGoogle Scholar
  10. 10.
    Piddock LJ (2006) Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin Microbiol Rev 19:382–402CrossRefPubMedGoogle Scholar
  11. 11.
    Lomovskaya O, Bostian KA (2006) Practical applications and feasibility of efflux pump inhibitors in the clinic – a vision for applied use. Biochem Pharmacol 71:910–918CrossRefPubMedGoogle Scholar
  12. 12.
    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:e365CrossRefPubMedGoogle Scholar
  13. 13.
    Baquero F (2001) Low-level antibacterial resistance: a gateway to clinical resistance. Drug Resist Updat 4:93–105CrossRefPubMedGoogle Scholar
  14. 14.
    Martinez JL, Baquero F, Andersson DI (2007) Predicting antibiotic resistance. Nat Rev Microbiol 5:958–965CrossRefPubMedGoogle Scholar
  15. 15.
    Lomovskaya O, Zgurskaya HI, Totrov M, Watkins WJ (2007) Waltzing transporters and “the dance macabre” between humans and bacteria. Nat Rev Drug Discov 6:56–65CrossRefPubMedGoogle Scholar
  16. 16.
    Piddock LJ (2006) Multidrug-resistance efflux pumps – not just for resistance. Nat Rev Microbiol 4:629–636CrossRefPubMedGoogle Scholar
  17. 17.
    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 31:458–462CrossRefPubMedGoogle Scholar
  18. 18.
    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 61:1076–1082CrossRefPubMedGoogle Scholar
  19. 19.
    Couto I, Costa SS, Viveiros M, Martins M, Amaral L (2008) Efflux-mediated response of Staphylococcus aureus exposed to ethidium bromide. J Antimicrob Chemother 62:04–513CrossRefPubMedGoogle Scholar
  20. 20.
    Greulich KO (2004) Single molecule techniques for biomedicine and pharmacology. Curr Pharm Biotechnol 5:243–259CrossRefPubMedGoogle Scholar
  21. 21.
    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 28:639–653CrossRefPubMedGoogle Scholar
  22. 22.
    Hendrikse NH (2000) Monitoring interactions at ATP-dependent drug efflux pumps. Curr Pharm Des 6:1653–1668CrossRefPubMedGoogle Scholar
  23. 23.
    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 59:1261–1264CrossRefPubMedGoogle Scholar
  24. 24.
    Shapiro HM (2008) Flow cytometry of bacterial membrane potential and permeability. Methods Mol Med 142:175–186CrossRefPubMedGoogle Scholar
  25. 25.
    Aszalos A (2007) Drug-drug interactions affected by the transporter protein, P-glycoprotein (ABCB1, MDR1) I. Preclinical aspects. Drug Discov Today 12:833–837CrossRefPubMedGoogle Scholar
  26. 26.
    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 7:576–586CrossRefPubMedGoogle Scholar
  27. 27.
    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 10:1409–1422CrossRefGoogle Scholar
  28. 28.
    Joux F, Lebaron P (2000) Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes Infect 2:1523–1535CrossRefPubMedGoogle Scholar
  29. 29.
    Jernaes MW, Steen HB (1994) Staining of Escherichia coli for flow cytometry: influx and efflux of ethidium bromide. Cytometry 17:302–309CrossRefPubMedGoogle Scholar
  30. 30.
    Duffy L, Dykes GA (2006) Growth temperature of four Campylobacter jejuni strains influences their subsequent survival in food and water. Lett Appl Microbiol 43:596–601CrossRefPubMedGoogle Scholar
  31. 31.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, USAGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Miguel Viveiros
    • 1
  • Liliana Rodrigues
    • 2
  • Marta Martins
    • 2
  • Isabel Couto
    • 1
    • 3
  • Gabriella Spengler
    • 2
  • Ana Martins
    • 2
  • Leonard Amaral
    • 1
    Email author
  1. 1.Unit of Mycobacteriology, Instituto de Higiene e Medicina TropicalUniversidade Nova de Lisboa (IHMT/UNL)LisboaPortugal
  2. 2.Unit of Mycobacteriology and UPMM, Instituto de Higiene e Medicina TropicalUniversidade Nova de Lisboa (IHMT/UNL)LisboaPortugal
  3. 3.Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e TecnologiaUniversidade Nova de LisboaCaparicaPortugal

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