Fluorescence enlightens RND pump activity and the intrabacterial concentration of antibiotics

To understand antibiotic resistance in Gram-negative bacteria, a key point is to investigate antibiotic accumulation, which is defined by influx and efflux. Several methods exist to evaluate membrane permeability and efflux pump activity, but they present disadvantages and limitations. An optimized spectrofluorimetric method using intrinsic tryptophan fluorescence as an internal standard, as well as a complementary microfluorimetric assay following time-course accumulation in intact individual cells, have been developed. Comparing the latter population and single cell approaches can lead to an understanding of phenotypic heterogeneity within a population. The two methodologies lead to determination of parameters, concentration, accumulation rates and localization that contribute to emerging concepts (RTC2T, SICAR) with the aim of identifying and detailing antibiotic chemotypes involved in influx/efflux.