Spectrofluorimetric quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial membranes

Abstract

The efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. This is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. Precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMSF). These different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (OM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. The three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (WCLs) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d.

The challenge of intracellular antibiotic accumulation, a function of fluoroquinolone influx versus bacterial efflux

“…These compounds differ in their molecular and physicochemical properties while they share a similar core structure (Table 1). Susceptibility assays were performed using isogenic E. coli K12 strains expressing various levels of AcrAB: AG100 (wild type), AG100A (AcrAB-efficient mutant), and AG102 (overexpressing AcrAB) 27 (see "Methods").…”

“…However, the ability of such compounds to reach effective cytoplasmic concentrations remains a key factor for their antimicrobial activity, a question that has not been properly addressed until now. The Innovative Medicine Initiative programs (www.imi.europa.eu) have supported the development of appropriate methods and concepts to measure translocation and to quantify the intrabacterial concentration of drugs 9,27 . Using fluorimetric assays, we previously showed that the molecular properties of three FQs dictate their capacity to accumulate in E. coli expressing or not the major multidrug efflux transporter AcrB, in complex with the adapter protein AcrA (in the following denoted as AcrAB) 8,27 .…”

“…The Innovative Medicine Initiative programs (www.imi.europa.eu) have supported the development of appropriate methods and concepts to measure translocation and to quantify the intrabacterial concentration of drugs 9,27 . Using fluorimetric assays, we previously showed that the molecular properties of three FQs dictate their capacity to accumulate in E. coli expressing or not the major multidrug efflux transporter AcrB, in complex with the adapter protein AcrA (in the following denoted as AcrAB) 8,27 . The SICAR (Structure Intracellular Concentration Activity Relationship) index has been proposed for quantifying accumulation of FQs and other antibiotics 8,10,12,21,22 .…”

Antibiotic transport kinetics in Gram-negative bacteria revealed via single-cell uptake analysis and mathematical modelling

“…Further, unlike previous studies using deep UV illumination to study antibiotic uptake in single 20 cells (11,21), since our excitation wavelength is 365 nm, we can work with standard optics and light sources, rather than needing quartz objectives and cover slips, and deep UV light sources which may not be easily accessible.…”

“…These mechanisms 10 are further complicated by the fact that the expression and activity of porins and efflux pumps vary i) with the microenvironment conditions (8) and ii) within an isogenic population exposed to the same environmental landscape (9). Many existing techniques suffer from the requirement of complex washing steps (3,10), with cells only studied after resuspension in contrived nutrient environments (11,12); the washes also increase the chance of cell lysis and efflux or diffusion of 15 the analyte from the cells, besides affecting cellular physiology. Furthermore, the most commonly used techniques are population level assays which cannot investigate heterogeneity in uptake at the single-cell or at the subcellular level.…”