Intracellular Quantification of an Antibiotic Metal Complex in Single Cells of <i>Escherichia coli</i> Using Cryo-X-ray Fluorescence Nanoimaging

Bacterial resistance is a major public health challenge. In Gram-negative bacteria, the synergy between multidrug efflux pumps and outer membrane impermeability determines the intracellular concentration of antibiotics. Consequently, it also dictates antibiotic activity on their respective targets. Previous research has employed spectrofluorimetry and synchrotron radiation-based DUV microscopy as tools for monitoring the accumulation of fluoroquinolone antibiotics in bacteria at population and single-cell scales, respectively. Here, we show that cryo-XRF nanoimaging allows intracellular localization and quantification of a fluoroquinolone metal complex accumulation in <i>Escherichia coli</i> with different efflux pump expression levels. This method offers a promising avenue for elucidating the intracellular behavior of a range of metallodrugs in bacteria and for designing novel agents with unique mechanisms of action.