Sensing Living Bacteria in Vivo Using d-Alanine-Derived ¹¹C Radiotracers

Incorporation of d-amino acids into peptidoglycan is a unique metabolic feature of bacteria. Since d-amino acids are not metabolic substrates in most mammalian tissues, this difference can be exploited to detect living bacteria in vivo. Given the prevalence of d-alanine in peptidoglycan muropeptides, as well as its role in several antibiotic mechanisms, we targeted this amino acid for positron emission tomography (PET) radiotracer development. d-[3-¹¹C]Alanine and the dipeptide d-[3-¹¹C]alanyl-d-alanine were synthesized via asymmetric alkylation of glycine-derived Schiff-base precursors with [¹¹C]methyl iodide in the presence of a cinchonidinium phase-transfer catalyst. In cell experiments, both tracers showed accumulation by a wide variety of both Gram-positive and Gram-negative pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. In a mouse model of acute bacterial myositis, d-[3-¹¹C]alanine was accumulated by living microorganisms but was not taken up in areas of sterile inflammation. When compared to existing clinical nuclear imaging tools, specifically 2-deoxy-2-[¹⁸F]fluoro-d-glucose and a gallium citrate radiotracer, d-alanine showed more bacteria-specific uptake. Decreased d-[3-¹¹C]alanine uptake was also observed in antibiotic-sensitive microbes after antimicrobial therapy, when compared to that in resistant organisms. Finally, prominent uptake of d-[3-¹¹C]alanine uptake was seen in rodent models of discitis-osteomyelitis and P. aeruginosa pneumonia. These data provide strong justification for clinical translation of d-[3-¹¹C]alanine to address a number of important human infections.