Abstract
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Objectives: The clinical need for timely diagnosis of life-threatening bacterial infections especially in immune-compromised patients has led to novel imaging approaches for pathogen specific detection of infections. This includes positron emission tomography (PET) with D-amino acids based radiotracers, which unlike mammalian hosts, are primarily incorporated by bacteria for peptidoglycan synthesis. Glutamine is essential for varied physiological processes governing bacterial survival, growth, biofilm formation, and virulence. Our proof-of-concept study compared D-[5-11C]-Glutamine with its L-isomer to develop an efficient PET imaging method targeting bacterial soft tissue infections. The study used dual infection myositis mouse models of Gram-positive (methicillin resistant Staphylococcus aureus (MRSA)) and Gram-negative (Escherichia coli (E. coli)) bacteria. Methods: We performed in vitro comparative studies between D versus L-[5-11C]-Glutamine evaluating time-dependent uptake and specificity in MRSA and E. coli, and mammalian HEK293 cells (uptake study), along with sensitivity evaluation across a bacterial panel. Comparative PET imaging studies were performed in neutropenic CD-1 mice, by injecting MRSA and E. coli in individual hind-limbs, and heat-killed bacteria in the shoulder muscle to induce localized sterile inflammation. Mice were injected with 8.7 - 13.4 µCi/g of each tracer and sequentially scanned by PET from 20 - 40 min post-injection followed by computed tomography. Ex vivo analyses included histological staining and microbiological culture assessments. Results and Discussion: Comparative in vitro assessments between the tracers showed ~10 - 12-fold higher bacterial uptake of L versus D-[5-11C]-Glutamine. However, rates of uptake increase from 30 - 90 min incubation were significantly higher for D versus L-[5-11C]-Glutamine in E. coli (p < 0.0001) and MRSA (p < 0.05). This may imply improved infection-to-background contrast at later imaging time-points with D-[5-11C]-Glutamine, which is desirable. No substantial uptake was observed in heat-killed cultures. Mammalian cells also showed incremental uptake from 30 to 90 min incubation (~8-fold, p < 0.0001) for L-[5-11C]-Glutamine, whereas no substantial change in D-[5-11C]-Glutamine uptake was observed during this time. These data confirmed our rationale for differential bacterial versus host utilization of D-[5-11C]-Glutamine. In vivo studies demonstrated substantially higher L-[5-11C]-Glutamine uptake than D-[5-11C]-Glutamine in most organs, except the kidneys, indicating increased renal excretion of D-[5-11C]-Glutamine by hosts due to non-utilization. Remarkably, PET imaging with D-[5-11C]-Glutamine resulted in significantly higher infection-to-background contrast ratios than L-[5-11C]-Glutamine (in E. coli: 1.64; in MRSA: 2.60, p = 0.0004, Figure 1). Moreover, lack of significant uptake in sterile inflammation sites (heat-killed site/muscle: ~1.04) confirmed the infection targeting specificity of D-[5-11C]-Glutamine.
Conclusions: Our findings indicate that D-[5-11C]-Glutamine has clinical potential for PET imaging of bacterial infections by specifically targeting active bacterial processes. Acknowledgement: This work was partially supported by the Cancer Prevention and Research Institute of Texas (CPRIT RP1706638 & RP110771 X.S.), the Dr. Jack Krohmer Professorship Funds, and the National Heart, Lung and Blood Institute (NIH T32 HL134613). Figure 1. PET Imaging with L and D-[5-11C]-Glutamine before (baseline) and after bacterial infection in mice