Fig. 8

Evaluation of efflux in resistant isolates. A Qualitative detection of EtBr efflux by employing the EtBr agar cartwheel method. Isolates were swabbed on tryptone soya agar plates, containing different concentrations of EtBr under UV transilluminator showing positive fluorescence growth (negative efflux) (c, e), negative fluorescence growth (positive efflux) (a, b and d) and intermediate efflux (f). B Quantitative fluorometric assay of EtBr efflux was performed for selected resistant isolates with MICs to biocides > MIC₅₀ or < MIC₅₀. The efflux assays were done under conditions that grant EtBr maximum accumulation in the presence of the efflux pump inhibitor dinitrophenol and limited energy supply (absence of glucose and low temperature). C The EtBr efflux is presented in terms of relative fluorescence (RF). All fluorescence measurements were done at wavelengths for EtBr 530 nm for excitation and 585 nm for emission. All data were acquired at 25 °C in cycles of 60 s, for 1 h time. Each test was repeated in triplicates and the obtained results were averaged. The RF for each tested isolate (MICs to biocides > MIC₅₀ or < MIC₅₀) was calculated; and results were expressed as means ± SD, p value < 0.05 was considered significant employing Student’s t-test. The efflux was significantly increased in tolerant isolates with MIC ≥ MIC₅₀. This indicates that the efflux plays an important role in conferring cross-resistance to both biocides and MLS antibiotics