Rapid profiling of polymyxin B susceptibility in Gram-negative bacteria with a novel polymyxin-derived fluorescent probe

• We designed and synthesized a fluorescent PMX-NBD probe via ‘click’ chemistry. • The modified PMX derivatives retained similar antimicrobial activity to the parent PMXB. • PMX-NBD showed its ability to label the membrane of Gram-negative bacteria. • A flow-cytometry method has been developed to quickly determine bacterial susceptibility to polymyxin B within 45 min. • It is a promising method to determine the resistance profile of bacterial isolates. Polymyxins are considered last-resort antibiotics for treating Gram-negative bacterial infections. The alarming global rise in polymyxin-resistant bacteria coupled with the slowness of conventional antimicrobial susceptibility testing methods leads to an unmet need for rapid susceptibility testing to facilitate timely and targeted therapeutic interventions. In this study, we aimed to develop novel polymyxin-derived fluorescent probes and improve the identification of polymyxin resistance by using these pathway-specific tools. We designed, synthesized, and profiled a polymyxin-fluorophore derivative (PMX-NBD) with the small fluorophore NBD attached the central cyclic core of polymyxin via a triazole linker. We then applied PMX-NBD to develop RAPID FC , a flow cytometry method that can rapidly and accurately profile polymyxin susceptibility of various Gram-negative pathogens. In contrast to many previously reported polymyxin probes, the fluorescent PMX-NBD retains the antimicrobial activity and profile of the parent polymyxin. Furthermore, we tested probe uptake in 49 strains encompassing E. coli , K. pneumoniae , P. aeruginosa , A. baumannii and S. aureus with a range of polymyxin sensitivity, leading to a susceptibility testing method RAPID FC . Susceptibility to PMXB was defined by the difference between the Geometric Mean (GMean) of PMX-NBD fluorescence intensity at the two concentrations, with sensitive strains showing a greater variation compared to resistant strains. The RAPID FC method is easy to implement and only requires 45 min compared to the broth microdilution method, which requires at least 24 h. However, the standard protocol failed to provide clear discrimination for A. baumannii , a key WHO-priority pathogen, necessitating a modified, species-specific approach for this organism. The RAPID FC method, based on the polymyxin-derived fluorescent probe, is fast and applicable to multiple species, making it a promising antimicrobial susceptibility method to guide appropriate therapy in the clinic. The case of A. baumannii demonstrates that species-specific resistance mechanisms may require tailored optimization of detection protocols.