Research
Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli
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* Corresponding author: Miguel Viveiros mviveiros@ihmt.unl.pt
1 Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Rua da Junqueira 100, 1349-008 Lisboa, Portugal
2 UPMM, IHMT/UNL, Rua da Junqueira 100, 1349-008 Lisboa, Portugal
3 Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, 2829-516 Caparica, Portugal
4 IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Av Rovisco Pais, 1049-001 Lisboa, Portugal
5 Instituto de Patologia e Imunologia Molecular, Universidade do Porto, Rua Dr Roberto Frias, 4200-465 Porto, Portugal
6 Cost Action BM0701 (ATENS)
Journal of Biological Engineering 2009, 3:18 doi:10.1186/1754-1611-3-18
Published: 16 October 2009Abstract
Background
Efflux pump activity has been associated with multidrug resistance phenotypes in bacteria, compromising the effectiveness of antimicrobial therapy. The development of methods for the early detection and quantification of drug transport across the bacterial cell wall is a tool essential to understand and overcome this type of drug resistance mechanism. This approach was developed to study the transport of the efflux pump substrate ethidium bromide (EtBr) across the cell envelope of Escherichia coli K-12 and derivatives, differing in the expression of their efflux systems.
Results
EtBr transport across the cell envelope of E. coli K-12 and derivatives was analysed by a semi-automated fluorometric method. Accumulation
and efflux of EtBr was studied under limiting energy supply (absence of glucose and
low temperature) and in the presence and absence of the efflux pump inhibitor, chlorpromazine.
The bulk fluorescence variations were also observed by single-cell flow cytometry
analysis, revealing that once inside the cells, leakage of EtBr does not occur and
that efflux is mediated by active transport. The importance of AcrAB-TolC, the main
efflux system of E. coli, in the extrusion of EtBr was evidenced by comparing strains with different levels
of AcrAB expression. An experimental model was developed to describe the transport
kinetics in the three strains. The model integrates passive entry (influx) and active
efflux of EtBr, and discriminates different degrees of efflux between the studied
strains that vary in the activity of their efflux systems, as evident from the calculated
efflux rates: 
= 0.0173 ± 0.0057 min-1; 
= 0.0106 ± 0.0033 min-1; and 
= 0.0230 ± 0.0075 min-1.
Conclusion
The combined use of a semi-automated fluorometric method and an experimental model allowed quantifying EtBr transport in E. coli strains that differ in their overall efflux activity. This methodology can be used for the early detection of differences in the drug efflux capacity in bacteria accounting for antibiotic resistance, as well as for expedite screening of new drug efflux inhibitors libraries and transport studies across the bacterial cell wall.