[(18)F]FDG-6-P as a novel in vivo tool for imaging staphylococcal infections

Abstract

Background: Management of infection is a major clinical problem. Staphylococcus aureus is a Gram-positive bacterium which colonises approximately one third of the adult human population. Staphylococcal infections can be life-threatening and are frequently complicated by multi-antibiotic resistant strains including methicillin-resistant S. aureus (MRSA). Fluorodeoxyglucose ([(18)F]FDG) imaging has been used to identify infection sites; however, it is unable to distinguish between sterile inflammation and bacterial load. We have modified [(18)F]FDG by phosphorylation, producing [(18)F]FDG-6-P to facilitate specific uptake and accumulation by S. aureus through hexose phosphate transporters, which are not present in mammalian cell membranes. This approach leads to the specific uptake of the radiopharmaceutical into the bacteria and not the sites of sterile inflammation.

Methods: [(18)F]FDG-6-P was synthesised from [(18)F]FDG. Yield, purity and stability were confirmed by RP-HPLC and iTLC. The specificity of [(18)F]FDG-6-P for the bacterial universal hexose phosphate transporter (UHPT) was confirmed with S. aureus and mammalian cell assays in vitro. Whole body biodistribution and accumulation of [(18)F]FDG-6-P at the sites of bioluminescent staphylococcal infection were established in a murine foreign body infection model.

Results: In vitro validation assays demonstrated that [(18)F]FDG-6-P was stable and specifically transported into S. aureus but not mammalian cells. [(18)F]FDG-6-P was elevated at the sites of S. aureus infection in vivo compared to uninfected controls; however, the increase in signal was not significant and unexpectedly, the whole-body biodistribution of [(18)F]FDG-6-P was similar to that of [(18)F]FDG.

Conclusions: Despite conclusive in vitro validation, [(18)F]FDG-6-P did not behave as predicted in vivo. However at the site of known infection, [(18)F]FDG-6-P levels were elevated compared with uninfected controls, providing a higher signal-to-noise ratio. The bacterial UHPT can transport hexose phosphates other than glucose, and therefore alternative sugars may show differential biodistribution and provide a means for specific bacterial detection.

Keywords: Infection diagnosis; NanoPET-CT imaging; Pre-clinical; S. aureus.

Figure 1

Determination of the purity of [ ¹⁸ F]FDG-6-P. By (a) iTLC at 0 h and (b) RP-HPLC after incubation of the radiopharmaceutical in water for 3 h. The retention profiles of [¹⁸F]FDG-6-P were easily resolved from that of [¹⁸F]FDG, the most likely breakdown product of [¹⁸F]FDG-6-P. No changes in the [¹⁸F]FDG-6-P peak or retention time were observed.

Figure 2

Uptake of [ ¹⁸ F]FDG-6-P and [ ¹⁸ F]FDG by S. aureus and mammalian cells. (a) S. aureus RN6390 and the isogenic UHPT-deficient mutant were incubated with either [¹⁸F]FDG or [¹⁸F]FDG-6-P. The activity associated with each of the strains after incubation with [¹⁸F]FDG (black) or [¹⁸F]FDG-6-P (white) was measured using a gamma counter. Counts were normalised to remove ‘no cell’ control counts and to compare [¹⁸F]FDG-6-P uptake with [¹⁸F]FDG uptake. Error bars show SEM. **P = 0.0015; ****P < 0.0001. Data were collected from three independent repeats. (b) The activity associated with mammalian cell lines HL60, Jurkat, AGS, THP1, HIB-1B and 3 T3-L1 after incubation with [¹⁸F]FDG (black) or [¹⁸F]FDG-6-P (white) was measured using a gamma counter. The counts were normalised to compare [¹⁸F]FDG-6-P uptake with [¹⁸F]FDG uptake. Error bars show SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Differences in the means of cells incubated with [¹⁸F]FDG-6-P were not significant.

Figure 3

In vivo biodistribution of [ ¹⁸ F]FDG-6-P and [ ¹⁸ F]FDG. (a) Representative optical image confirming the presence of BL catheter-associated S. aureus on the day of the nanoScan PET-CT scan. (b) Whole body biodistributions from nanoPET-CT scans of mice with sc implanted catheters inoculated with or without S. aureus 1 h after ip injection of approximately 10 MBq [¹⁸F]FDG or [¹⁸F]FDG-6-P; catheters are shown with arrows. SUVs were calculated from 3D ROIs drawn for each mouse from the whole-body nanoScan PET-CT images for (c) infected ([¹⁸F]FDG n = 3; [¹⁸F]FDG-6-P n = 3) and (d) uninfected mice ([¹⁸F]FDG n = 3; [¹⁸F]FDG-6-P n = 2. Black circle, [¹⁸F]FDG; black square, [¹⁸F]FDG-6-P). Bars on graph show median. BAT, brown adipose tissue.

Figure 4

Stability of [ ¹⁸ F]FDG-6-P in the blood. Blood extracted from mice was incubated with [¹⁸F]FDG or [¹⁸F]FDG-6-P for 1 h. The blood was analysed by iTLC to determine whether any additional peaks indicating dephosphorylation of [¹⁸F]FDG-6-P. The dotted line shows the peak for the [¹⁸F]FDG-6-P standard, and the dashed line shows the peak for the [¹⁸F]FDG standards which were not incubated with blood.

Figure 5

Accumulation of [ ¹⁸ F]FDG and [ ¹⁸ F]FDG-6-P at the catheter infection site. Mice with S. aureus infections (or uninfected control mice) were injected with approximately 10 MBq [¹⁸F]FDG (n = 3 infected, n = 3 uninfected) or approximately 10 MBq [¹⁸F]FDG-6-P (n = 3 infected, n = 2 uninfected) 1 h prior to nanoScan PET-CT imaging. (a) Representative 3D colour map of catheter regions cropped from whole-body nanoScan PET-CT images. Images are shown with and without CT. (b) SUV values were calculated for S. aureus-infected (black circle) and uninfected mice (black square). Mann-Whitney U tests confirmed that there were no significant differences in SUVs between infected and uninfected mice injected with either [¹⁸F]FDG (P = 0.7619) or [¹⁸F]FDG-6-P (P = 0.0556). Bars on the graph show median SUVs for each group (n = 3 for [¹⁸F]FDG infected and n = 3 for uninfected mice; n = 3 for [¹⁸F]FDG-6-P infected mice and n = 2 uninfected mice). (c) The infected (I) to uninfected (UI) ratio (I/UI) for the catheter sites of mice injected with [¹⁸F]FDG and [¹⁸F]FDG-6-P was calculated by dividing the mean infected catheter SUV by the mean uninfected catheter SUV for each cohort of mice.