PET imaging of bacterial infections with fluorine-18-labeled maltohexaose

Abstract

A positron emission tomography (PET) tracer composed of (18)F-labeled maltohexaose (MH(18)F) can image bacteria in vivo with a sensitivity and specificity that are orders of magnitude higher than those of fluorodeoxyglucose ((18)FDG). MH(18)F can detect early-stage infections composed of as few as 10(5) E. coli colony-forming units (CFUs), and can identify drug resistance in bacteria in vivo. MH(18)F has the potential to improve the diagnosis of bacterial infections given its unique combination of high specificity and sensitivity for bacteria.

Keywords: bacterial infections; maltodextrin transporters; maltohexaose; positron emission tomography; radiochemistry.

Figure 1

MH¹⁹F has high specificity for bacteria and is robustly internalized by bacteria. a, MH¹⁹F has high specificity for bacteria over hepatocytes. E. coli (EC), EC with LamB mutation (LamB) and mammalian cells were incubated with 500 μM MH¹⁹F for 1 hour in the presence or absence of 50 mM maltohexaose (MH). The intracellular MH¹⁹F concentration was determined and normalized to protein content. Bacteria robustly accumulate MH¹⁹F whereas hepatocytes have negligible uptake. The uptake of MH¹⁹F in EC is inhibited by a large excess of maltohexaose, and the uptake of MH¹⁹F in LamB mutants is significantly reduced. The results are expressed as mean micromoles per gram of protein ± s.e.m. for n = 3 per group. b, The accumulation of MH¹⁹F in EC reaches millimolar concentrations. EC were incubated with 500 μM MH¹⁹F, and the intracellular concentration of MH¹⁹F was determined at different time points, n = 3 per group.

Figure 2

In vivo PET imaging of rats infected with E.coli (10⁷CFUs). a. Rats were infected in the left triceps muscle with 10⁷ E.coli, injected with MH¹⁸F, and dynamic PET scans were performed for 90 minutes using a microPET/CT. Infected muscles can be easily visualized after 90 min. b. Time activity curves of decay-corrected MH¹⁸F activity in the infected rat, generated from Figure 2a. Infected muscle has an 8.5 fold increase in radioactivity over PBS injected muscle. Arrows indicate the location of infected muscle (EC), PBS injected muscle (PBS) and healthy tissue (HT).

Figure 3

MH¹⁸F can detect as few as 10⁵ CFUs of E.coli (EC) in muscle infections. a1, MH¹⁸F can detect 10⁷ E.coli CFUs in rats. Rats were infected with 10⁷ E.coli and imaged with MH¹⁸F using a microPET/CT. The rat image is a representative result of four experiments, and identifies the infection site. a2, MH¹⁸F generates a 6 fold increase in radioactivity in infected muscles. b1, MH¹⁸F can detect as few as 10⁵ E.coli in rats. Rats were infected with 10⁵ E.coli CFUs and imaged with MH¹⁸F using a microPET/CT. The rat image is a representative result of four experiments, and identifies the infection site. b2, MH¹⁸F generates a 2.7 fold increase in radioactivity in infected muscles. Regions of interest (ROIs) including the infected muscles (target) or PBS injection areas (control) and healthy tissues (background) were identified and integrated using ASI Pro VM™ micro PET analysis software. The results in a2 and b2 are expressed as the target or control to background ratio (ROI ratio) ± s.e.m. for n = 4 per group. The ROI ratio is defined as the mean radioactivity in the target/the mean radioactivity in the background. The statistical significances in a2 and b2 were determined using a two-sample Student's t-test (*p ≤ 0.05 and ***p ≤ 0.001).

Figure 4

MH¹⁸F is more effective than ¹⁸FDG at imaging bacterial infections. A biodistribution study was performed with either MH¹⁸F or ¹⁸FDG in rats infected with 10⁹ E.coli CFUs. MH¹⁸F is efficiently cleared from un-infected tissues, whereas ¹⁸FDG has significant accumulation within the major organs. The results are expressed as % injected dose/gram tissue ± s.e.m. for n = 4 per group. Statistical significance was determined using a two-sample Student's t-test (*p ≤ 0.05 and ***p ≤ 0.001).

Figure 5

MH¹⁸F can distinguish between live versus dead bacteria and can discriminate infections from inflammation. a1, MH¹⁸F can distinguish between live versus dead bacteria in vivo. Rats were infected with 10⁹ live and dead E.coli and imaged with MH¹⁸F using a microPET/CT. The rat image is a representative result of four experiments, and demonstrates that MH¹⁸F does not accumulate in dead bacteria. a2, E.coli infected tissues had a 7 fold increase in radioactivity over muscles treated with dead bacteria. b1, ¹⁸FDG cannot distinguish between live and dead E.coli infected tissues. Rats were infected with 10⁹ live and dead E.coli CFUs and imaged with ¹⁸FDG using a microPET/CT. The image is a representative result of four experiments, and demonstrates that ¹⁸FDG cannot discriminate live bacteria from dead bacteria. b2, ¹⁸FDG accumulates in both live and dead bacteria infected tissues. ROIs including the infected muscles (target) and healthy tissues (background) from a1 and b1were identified and integrated using ASI Pro VM™ micro PET analysis software. The results in a2 and b2 are expressed as ROI ratio ± s.e.m. for n = 4 per group. The ROI ratio is defined as the mean radioactivity in the target/the mean radioactivity in the background. The statistical significance in a2 was determined using a two-sample Student's t-test (***p ≤ 0.001).

Figure 6

MH¹⁸F can measure drug resistance and monitor the therapeutic effect of antibiotics in vivo. a1, MH¹⁸F can identify drug resistance in bacteria in vivo. Rats were infected with 10⁹ CFUs of ampicillin-resistant E.coli (DR EC) and wild-type E.coli (EC), treated with ampicillin and imaged with MH¹⁸F using a microPET/CT. The rat image is a representative result of four experiments, and demonstrates that MH¹⁸F only accumulates in DR EC infected muscles. a2, DR EC generated a 10 fold increase in radioactivity over EC. b1, MH¹⁸F can monitor the therapeutic effect of antibiotics. Rats were infected with DR EC and EC, treated with ciprofloxacin and imaged with MH¹⁸F using a microPET/CT. The rat image is a representative result of four experiments, and demonstrates that both DR EC and EC infected muscles have weak accumulation of MH¹⁸F. a2, Both infected tissues have weak radioactivity. ROIs including the infected muscles (target) and healthy tissues (background) from a1 and b1 were identified and integrated using ASI Pro VM™ micro PET analysis software. The results in a2 and b2 are expressed as ROI ratio ± s.e.m. for n = 4 per group. The ROI ratio is defined as the mean radioactivity in the target/the mean radioactivity in the background. The statistical significance in a2 was determined using a two-sample Student's t-test (***p ≤ 0.001).

Scheme 1

Synthesis of MH¹⁸F. MH¹⁸F is composed of ¹⁸F-fluoride conjugated to maltohexaose and was synthesized by one-step nucleophilic ¹⁸F-fluorination of brosylate-maltohexaose 3.