ABSTRACT: Bacterial infections are a major threat to human health. Many bacteria, particularly Gram-positives, form difficult- to-eradicate biofilms on catheters, cardiac devices and bone implants such as joint replacements. Because of the difficulty in treating established biofilm infections, early detection is advantageous. However, current diagnostic imaging modalities rely on nonspecific host features such as inflammation and edema, rather than detecting the bacteria themselves. The antibiotic vancomycin binds with high affinity and specificity to Gram- positive bacteria, and antibiotic-derivatives have been explored as imaging agents. In this application, we conjugate vancomycin to the siderophore DFOB, which binds with nM affinity to the virulence-essential surface displayed lipoprotein receptor (FhuD2) on S. aureus and other Gram-positives, and is subsequently internalized. DFOB is also an FDA approved chelation agent that forms stable complexes with transition metal PET isotopes (68Ga, 89Zr, and 44Sc). In the first Specific Aim, we test the in vitro affinity and specificity of bivalent radiometal- labeled Vanco-PEG-DFOB on target proteins, on live planktonic bacteria, as well as in clinically relevant biofilm embedded systems. We will determine the role of PEG spacer and transition metal isotope on the binding capability, and compare with monovalent tracers. In the second aim, we test the contrast and detection capacity of Vanco-PEG-DFOB using in vivo models of implant infection for both in-dwelling catheters and periprosthetic joint surfaces. We compare this novel bivalent and internalizing strategy to control monovalent radiotracers, and the clinically applied 18F-FDG, to generate data that may motivate further translational development of Vanco- PEG-DFOB for delineation of sites of infection. Importantly, this discovery platform can be adapted for other microbe-binding compounds to expand utility to a wide variety of infections.