The global prevalence of antimicrobial resistance (AMR) seen in microbes is threatening human health (O’Neill, 2016). Fundamental research into how antibiotics work is critical to better understand their mechanism(s) of action, and how bacterial resistance develops. Our group has been developing a suite of antibiotic-derived fluorescent probes based on major classes of antibiotics to facilitate these types of studies. We now report a probe (green and blue variant) derived from daptomycin, a calcium-dependent intravenous antibiotic used for the treatment of bacteremia, right-sided endocarditis and complicated skin and skin structure infections (CSSSI) caused by Gram-positive bacteria. Daptomycin is active against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA) and vancomycin-resistant enterococci (VRE) which cause substantial global morbidity (Murray et al., 2022).
Minimum inhibitory concentration (MIC) testing, flow cytometry, confocal fluorescence microscopy and microfluidic assays were performed to characterise this novel, mechanism specific daptomycin probe for future use in qualitative and quantitative fluorescence analysis assays.
MIC results showed that the daptomycin probe maintained similar activity profiles to the parent antibiotic against daptomycin susceptible and resistant bacteria, including methicillin and vancomycin resistant strains. Flow cytometry data confirmed the probe’s selectivity for Gram-positive bacteria in a calcium dependent manner, with rapid staining (<30 min) across multiple concentrations; temperature variation appeared to have limited effect on binding. Confocal fluorescence microscopy showed binding of the probe to the outer membrane of S. aureus, with no uptake into the cytoplasm of cells.
It was concluded that this fluorescent daptomycin probe is a suitable model for daptomycin mode of action and resistance studies as it retains similar activity to the parent antibiotic. Future experiments will investigate further the effect of different concentrations and staining times using confocal microscopy, as well as co-staining using various other commercial probes to assess daptomycin activity and resistance mechanisms.