Summary

Bacteria from the Burkholderia cepacia complex (BCC) are generally considered to be non-pathogenic to the healthy population. However, some of these species may cause serious nosocomial infections in immunocompromised patients; as such, it is essential to diagnose these infections rapidly so that adequate treatment can be initiated.

BCC bacteria synthesize four different types of siderophores: pyochelin, cepabactin, cepaciachelin, and ornibactin (ORNB) under iron-limited conditions, e.g. during infectious processes that involve host−bacterial competition for iron. They play an important role in essential microbial metabolism, pathogenicity, virulence, and− in some cases−biofilm formation. Due to the similar physicochemical properties of iron and gallium, various siderophores can bind to gallium-68.

In this study, the use of gallium-68-labeled ORNB for positron emission tomography imaging is reported. Results show that in mice, the complex did not accumulate excessively in organs and was excreted in the urine. It was demonstrated that the [⁶⁸Ga]Ga-ORNB complex accumulates at the site of Burkholderia multivorans infection, including pneumonia, in two animal infection models. These results suggest that [⁶⁸Ga]Ga-ORNB is a promising tool for the diagnosis, monitoring, and evaluation of the therapeutic response to B. cepacia complex infection.

Results from nanoScan® PET/CT

Animal experiments were performed on female 8-10 week old Balb/c mice and female 8-10 week old Lewis rats.

The muscle infection model was performed on immunosuppressed and non-immunosuppressed mice. They were injected i.p. with cyclophosphamide five and three days prior to infection, as well as on the day of infection. On the day of infection, all mice were injected intramuscularly (i. m.) with 50 μL of bacterial culture containing BUMU (c = 104−108 cfu/ mL) into the muscle of the left hind leg. To test the specificity of the in vivo uptake of [⁶⁸Ga]Ga-ORNB, 50 μL of different bacterial cultures (BUMU or E. coli; live or heat-inactivated), turpentine oil (to right hind leg muscle. The microbial infections were allowed to develop for 5 min for the dynamic imaging study, for 45 min−48 h for the monitoring of [⁶⁸Ga]Ga-ORNB uptake, and for in vivo specificity testing. The induction of sterile inflammation lasted 24 h.

For the lung infection model, immunosuppressed rats were treated with cyclophosphamide five days and one day before infection. Rats were infected intratracheally with 100 μL of BUMU culture (c = 7−8 × 108 cfu/mL) and underwent PET/CT imaging 5−72 h after inoculation.

Animals under isoflurane anesthesia were r.o. injected with [⁶⁸Ga]Ga-ORNB (approximately ∼0.5 μg of ORNB) at a dose of 2−7 MBq per animal and placed in the Mediso NanoScan PET/CT imaging system for small animals (Mediso Medical Imaging Systems, Budapest, Hungary). After the administration of [⁶⁸Ga]Ga-ORNB, static imaging was initiated 30 and 90 min p.i. for non-infection imaging studies and 45 min p.i. for infection imaging studies. Dynamic imaging studies were started ∼5 min p.i. single FOV PET scans for mice and double FOV PET scans for rats were performed, followed by whole body helical CT scan. Image reconstruction was performed via Mediso Tera-Tomo 3D PET iterative reconstruction. The images were visualized, processed, and quantified in the Mediso InterView FUSION software. Quantitative analyses were performed on images of non-infected rats and rats with lung infections. The images were normalized to injected activity and animal weight. The results were expressed as percentage of injected dose per gram tissue (% ID/g).

• The biodistribution assays were performed using PET/CT imaging of non-infected mice injected with [⁶⁸Ga]Ga-ORNB. Results from the imaging demonstrated that [⁶⁸Ga]Ga-ORNB was rapidly cleared from the bloodstream, showed no accumulation in major organs and tissues, and was excreted via the kidneys. (Figure 4)

Figure 4. Maximum intensity projection (MIP) PET/CT images of in vivo [⁶⁸Ga ]Ga-ORNB biodistribution in normal mice 30 and 90 min after injection of [⁶⁸Ga ]Ga-ORNB.

• In a mouse model of BCC myositis, [⁶⁸Ga]Ga-ORNB accumulated in the infected left hind limb but not in the control-injected right hind limb. The signal observed in the infected limb was solely due to the underlying bacterial infection since no tracer accumulation was observed in the case of turpentine oil-induced myositis (Figure 5).

Figure 5. PET/CT in vivo imaging of [⁶⁸Ga ]Ga-ORNB biodistribution in a mouse model of BUMU infection in the left hind limb (red arrow) and various agents or microbial cultures in the right hind limb (white arrow): (1) saline, (2) turpentine oil, (3) heat-inactivated BUMU, and (4) E. coli. MIP images at 45 min after [⁶⁸Ga ]Ga-ORNB administration.

• The radioactive signal in the infected limb tended to decrease with lowest dose of BUMU detectable by PET imaging was 10⁵ cfu (Figure S7).

Figure S7: PET/CT in vivo imaging of [⁶⁸Ga ]Ga-ORNB biodistribution in the BUMU muscle infection model with various infectious doses in mice 5 h after infection and 45 min after [⁶⁸Ga ]Ga-ORNB administration (maximum intensity projections images). Yellow arrow indicates the infection.

• In the dynamic study, a radioactive signal could be detected in the infected hind limb as early as the first time frame (∼5 min post-injection and post-infection), and signal intensity in the limb increased across subsequent time frames (Figure S8).

Figure S8: PET in vivo dynamic study of [⁶⁸Ga ]Ga-ORNB biodistribution in the BUMU muscle infection model 5-90 min after infection and [⁶⁸Ga ]Ga-ORNB administration (maximum intensity projections images). H = heart, K = kidneys, B = bladder, INF = site of infection.

• In a rat model of pulmonary infection, [⁶⁸Ga]Ga-ORNB showed clear accumulation in the lungs of rats infected with BUMU. No radioactive signal was detected in the lungs of non-infected rats (Figure 6). A quantitative analysis revealed significant differences in maximal standardized uptake values (SUV_max) between non-infected and infected rats (0.88 ± 0.08 versus 7.17 ± 1.48; P < 0.01), as displayed in Figure 7.

Figure 6. PET/CT MIP images of [⁶⁸Ga ]Ga-ORNB in a control rat (1) and in a rat model of lung infection (BUMU) (2−5) 48−72 h after infection and 45 min after the injection of [⁶⁸Ga ]Ga-ORNB. Yellow arrows indicate the site of infection.

Figure 7. Comparison of radioactive signal uptake in the lungs of non-infected and infected rats (n = 4). Results are expressed as the maximal standardized uptake value (SUV_max); ***P < 0.01.

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