Caracterização e Modelação do Transporte de Brometo de Etídeo em Escherichia coli

Ana Laura Machado dos Santos Seara

Key information

Authors:

Ana Laura Machado dos Santos Seara (Ana Laura Machado dos Santos Seara Paixão)

Supervisors:

Miguel Viveiros Bettencourt (Miguel Viveiros Bettencourt); Gabriel António Amaro Monteiro (Gabriel A. Monteiro)

Published in

11/05/2007

Abstract

In this work we defined an automated method to demonstrate and quantify the intrinsic efflux pump activity in antibiotic susceptible strains and with a resistant multidrug phenotype. The protocol was defined having as standard strain the wild type E. coli k-12 AG100, which has its principal efflux system intact and functional - AcrAB-TolC. The method employs ethidium bromide (EtBr) as the efflux substrate and the fluorescence signal was monitorized by fluorimetry using the real-time thermocycler Rotor-Gene 3000TM. The method allowed us to analyze EtBr accumulation under limiting energy supply (absence of glucose; low temperature) as well as in the presence of efflux inhibitors. Data gathered showed that efflux is an active transport system mediated by efflux pumps. The role of AcrAB-TolC in the efflux of EtBr was highlighted by comparing strains which differ in efflux activity: AG100A (inactivated AcrAB-TolC) and AG100TET (over expression of AcrAB-TolC). The results suggested that EtBr might bind to several intracellular targets and once inside the cells there is no leaking out of EtBr. A mathematical model was developed to describe the transport kinetics in the three strains. The model shows that EtBr flows into the cell by passive diffusion. It allowed to discriminate the efflux between strains with different degrees of AcrAB-TolC activity (k-AG100= 0,0173 ± 0,0057 min-1; k-AG100A = 0,0106 ± 0,0033 min-1 e k-AG100TET = 0,0230 ± 0,0090 min-1). The model furthermore revealed the difference in permeability between AG100A and AG100 and suggested the existence of an inductive efflux mechanism in AG100TET.