Research

Fluorometric determination of ethidium bromide efflux kinetics in Escherichia coli

Laura Paixão¹ , Liliana Rodrigues^1,2 , Isabel Couto^1,3 , Marta Martins^1,2 , Pedro Fernandes⁴ , Carla CCR de Carvalho⁴ , Gabriel A Monteiro⁴ , Filipe Sansonetty⁵ , Leonard Amaral^1,2,6 and Miguel Viveiros^1,6

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)

author email corresponding author email

Journal of Biological Engineering 2009, 3:18doi:10.1186/1754-1611-3-18

Published:	16 October 2009

Abstract

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.