Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level

Abstract

Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and measurement of the crosstalk between fluorescence channels can provide real time quantification of drug. This technique represents a new method to assay drug translocation inside the cell and therefore incorporate rational drug design to impact antibiotic uptake.

A Gestalt approach to Gram-negative entry

“…As part of the IMI TRANSLOCATION project, Cinquin et al 84 use microspectrophotometric methods on single cells to measure translocation of certain drugs which have natural fluorescence excited by deep UV (DUV). This should be a useful, albeit low throughput, activity-independent approach to the problem.…”

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

“…(fluorescence emission between 330 and 360 nm) to obtain a fluorescence signal per bacterial cell 42 . Calibration curves were carried out to determine the quantity of molecules accumulated per cell: various concentrations of the studied FQ were mixed with bacterial lysates at OD 600 of 4.8 and measured using a spectrofluorimeter (n = 3 replicates) 27 .…”

Multidrug efflux pumps and their role in antibiotic and antiseptic resistance: a pharmacodynamic perspective

“…Moreover, this is often difficult to find relationship between MIC values and direct efflux measurements using fluorescent dyes substrate of efflux (e. g. ethidium bromide, 1,2ʹ -dinaphthylamine, Hoechst H33342). Advances in new noninvasive methodologies using fluorescence spectrometry associated to cell imaging by microscopy allow detecting and quantifying drugs inside individual living bacteria [100]. The significant obstacle to these methods is the interfering fluorescence of molecules used in the evaluation.…”