Discovery of a Thermostable Nigerose Phosphorylase for the Efficient Chemoenzymatic Radiosynthesis of a <i>S. aureus</i> –Targeted ¹⁸ F-Disaccharide

<i>Staphylococcus aureus</i> is a leading cause of life-threatening infections worldwide. The diagnosis and treatment of <i>S. aureus</i> infections are further complicated by the global rise of antimicrobial resistance. Therefore, rapid detection of active <i>S. aureus</i> remains a critical unmet need to provide effective infection management. In this study, we identified 2-deoxy-2-[¹⁸F]-fluorosakebiose ([¹⁸F]FSK) as an optimal radiotracer to detect active <i>S. aureus</i> and established its efficient chemoenzymatic radiosynthesis to facilitate clinical translation. <b>Methods</b>: Several [¹⁸F]FDG-derived disaccharides were obtained via reverse phosphorolysis: [¹⁸F]FSK (α-1,3–linked), 2-deoxy-[¹⁸F]-fluoromaltose (α-1,4–linked), 2-deoxy-2-[¹⁸F]-fluorolaminaribiose (β-1,3–linked), and 2-deoxy-2-[¹⁸F]-fluorocellobiose (β-1,4–linked). These tracers were screened in&nbsp;vitro in multiple <i>S. aureus</i> isolates to identify bacterial incorporation. The lead candidate, [¹⁸F]FSK, was further characterized via biodistribution and dosimetry analyses and evaluated in a <i>S. aureus</i> myositis model to assess antimicrobial treatment response. Finally, to promote the clinical translation of [¹⁸F]FSK, 2 different radiosynthetic strategies were investigated: reverse phosphorolysis of [¹⁸F]FDG using maltose phosphorylase and using newly identified nigerose (also called sakebiose) phosphorylases. <b>Results</b>: Nigerose phosphorylase–derived [¹⁸F]FSK was selected as the optimal radiotracer for detecting <i>S. aureus</i> because of its consistent and robust uptake in multiple <i>S. aureus</i> isolates. [¹⁸F]FSK demonstrated favorable distribution and elimination over time, with minimal nonspecific signals in uninfected organs. The estimated human effective doses indicated an effective dose comparable to that of [¹⁸F]FDG. The radiosynthesis of [¹⁸F]FSK, initially obtained as an accidental byproduct of maltose phosphorylase catalysis, was further improved using a nigerose phosphorylase originating from thermostable <i>Spirochaeta thermophila</i>, enabling nearly quantitative conversion of [¹⁸F]FDG to [¹⁸F]FSK (up to 97%). <b>Conclusion</b>: We demonstrated that [¹⁸F]FSK is a potent and robust PET radiotracer for the detection of active <i>S. aureus</i> in&nbsp;vivo and aids in the selection of an appropriate antimicrobial treatment. These findings highlight the potential use of [¹⁸F]FSK in promoting the effective management of <i>S. aureus</i> infections in clinical settings.