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

Bertrand Cinquin (1) , Laure Maigre (2) , Elizabeth Pinet (3) , Jacqueline Chevalier (3) , Robert A Stavenger (4) , Scott A Mills (5) , Matthieu Réfrégiers (1) , Jean-Marie Pagès (2)

1 SSOLEIL - Synchrotron SOLEIL
2 MCT - Membranes et cibles thérapeutiques
3 TMCD2 - Transporteurs membranaires, chimioresistance et drug-design
4 GlaxoSmithKline
5 AstraZeneca

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. using pre-synthesized folic acid, increasing the rate of folic acid synthesis). (iv) Changing the membrane permeability (e.g. downregulation of porins, overexpression of efflux pumps). Employing this last strategy, the bacterium is able to manage the intra-cellular concentration of antibiotics by modulating the entry or the ejection of active agents 7. Thereby, the effective concentration of drug is never reached inside the cell and consequently its activity is minimized. Furthermore, the relatively low concentration of antibiotic inside the bacterium can promote adaptation by developing the expression/selection of other resistance mechanisms 8,9. Thus, the " multi " in the term MDR can be read at different levels: multi because of the multiple antibiotic classes a bacterium can be resistant to, but also because multiple and various mechanisms contribute to the bacterial survival in the presence of antibacterial agents 10. Deciphering the biochemical basis and the mechanistic processes underlying the accumulation of antibacterial agents is essential to design and develop antibiotics that can achieve higher, more effective intracellular concentrations and avoid further spreading of resistance. This is particularly important with the continuing emergence and the worldwide distribution of MDR bacteria and the paucity of new antibacterial agents to treat MDR bacteria 11–13. The paper of 1 DISCO beamline,

Domaines: Sciences du Vivant [q-bio]

Bertrand Cinquin, Laure Maigre, Elizabeth Pinet, Jacqueline Chevalier, Robert A Stavenger, et al.. Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level. Scientific Reports, 2016, 5 (1), pp.17968 - 17968. ⟨10.1038/srep17968⟩. ⟨hal-01831684⟩