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

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. Multi-drug resistant (MDR) Gram-negative bacteria such as Escherichia coli (E. coli) and other Enterobacteriaceae are spreading rapidly and in many cases are capable of producing severe infections that can eventually lead to death (135 000 infections in Europe and 215 000 in the USA annually) and contributing to the concept of the ESKAPE alert in clinical bacteriology and emerging pathogens 1,2. The widespread use, and misuse, of antibiotics results in the generation/release of antibiotic concentration gradients in the environment, including humans, animals, water, etc 3,4. Consequently, bacteria are frequently exposed to subinhibitory concentrations of antibiotics leading to the evolution, selection and potential spreading of antibiotic resistance 3,5,6. Different resistance mechanisms have been highlighted and include: (i) drug inactivation or modification by enzymatic action (e.g. ß-lactamase, acetylase). (ii) Alteration/mutation or masking of targets (e.g. penicillin binding proteins, Type II topoisomerases). (iii) Alteration of metabolic pathways: bacteria resist antimicrobial agents by using alternate pathways than those inhibited by the molecule, or bacteria increase the production of the target metabolite (e.g.