Background: Bacteria are becoming resistant to every antibiotic, leading to multi-drug resistant ‘superbugs’. It is imperative to discover and develop new antibiotics to fight these superbugs, but for this to occur we require an improved understanding of how antibiotics work and how bacteria function and develop resistance. This requires new tools and techniques to advance our knowledge of bacterial metabolism, efflux pumps and other responses to antibiotics, allowing for analysis of key aspects of bacterial growth, division, metabolism and resistance. In order to create such tools, we have been systematically converting representatives of the major classes of antibiotics into mechanism-specific fluorescent probes that retain the biological profile of the parent compound.[1-4]
Materials/methods: Antibiotics are functionalised with an azide substituent in a position that minimises effects on antibiotic activity. These are then reacted by a facile dipolar cycloaddition with alkyne-substituted imaging components such as fluorophores or PET radioisotopes. The resulting adducts can be used as tools to image bacteria to understand antibiotic mechanisms of action and the development of resistance.
Results: We have successfully produced fluorescent probes based on glycopeptides (vancomycin), lipopeptides (polymyxin, daptomycin, octapeptins), oxazolidinones (linezolid), fluoroquinolones (ciprofloxacin), trimethoprim, antimicrobial peptides (tachyplesin, arenicin), macrolides (roxithromycin) and echinocandins, generally with retention of antimicrobial activity. Probes with intracellular targets have undergone single cell analysis in microfluidic devices, showing remarkable strain-dependent variation in heterogeneity in antibiotic uptake. The probes have been used to develop assays for antibiotic efflux and outer membrane permeabilization, to study localization of membrane-disrupting compounds, to interrogate antibiotic interactions with mammalian cells, and to develop PET imaging agents for infections.
Conclusions: Fluorescent probes derived from antibiotics are proving to be useful tools to visualize the interactions of antimicrobial compounds with microbes and human cells, and have been applied to develop new assays. We are keen to make this toolset widely available to the microbiology community to advance our understanding of antibiotic resistance.