Radiotracers for in situ infection imaging: Experimental considerations for in vitro microbial uptake of gallium-68-labeled siderophores

Abstract

In vitro screening of gallium-68(⁶⁸Ga)-siderophores in pathogens relevant to infections is valuable for determining species specificity, their effect on cell viability, and potential clinical applications. As the recognition and internalization of siderophores relies on the presence of receptor- and/or siderophore-binding proteins, the level of uptake can vary between species. Here, we report in vitro uptake validation in Escherichia coli with its native siderophore, enterobactin (ENT) ([⁶⁸Ga]Ga-ENT), considering different experimental factors. Compared with other reporting methods of uptake, '% Added dose/10⁹ CFU/mL (% AD/10⁹ CFU/mL),' considering the total viable count, showed a better comparison among microbial species. Later, in vitro screening with [⁶⁸Ga]Ga-desferrioxamine B (DFO-B) showed high uptake by Staphylococcus aureus and S. epidermidis; moderate uptake by Pseudomonas aeruginosa; poor uptake by E. coli, Candida albicans, and Aspergillus fumigatus; and no uptake by Enterococcus faecalis and C. glabrata. Except for S. epidermidis, [⁶⁸Ga]Ga-DFO-B did not reduce the cell viability.

Keywords: (68)Ga-siderophores; DFO-B; Diagnostics; Infection imaging; PET tracers; Uptake method.

Fig. 1

Validation of in vitro uptake of ⁶⁸Ga-siderophores in microbial cells. (A) Uptake of [⁶⁸Ga]Ga-ENT by E. coli expressed using different metrics. Uptake performed in early stationary phase E. coli cells after incubation with [⁶⁸Ga]Ga-ENT for 45 min at 37°C with shaking under iron-depleted and iron-replete (adding 10 µM FeCl₃) conditions. ‘% AD/10⁹ CFU/mL’ showed higher significance when compared between iron-depleted and iron-replete conditions considering the total viable counts. (B) Uptake of [⁶⁸Ga]Ga-TAFC by A. fumigatus in a 96-well filter plate. Uptake was performed by incubating 180 µL of mycelial cells with [⁶⁸Ga]Ga-TAFC for 45 min at 37°C with shaking under iron-depleted and iron-replete (30 µM FeCl₃) conditions. Error bars indicate standard deviations from three individual samples. The unpaired t-test for comparison among test groups showed significant differences (a P value of <0.05 expressed as ‘*’). [% AD: ‘% Added Dose’, considers activity in the cell pellets; ‘% AD/g of pellet’ considers the total weight of the pellets; ‘% AD/10⁹ CFU/mL’ expresses activity in the cell pellets in 10⁹ cells; % TA: ‘% Total Activity’, expresses activity there is in the cell pellets compared to the supernatant; DF: ‘Distribution ratio’, expresses the ratio of intracellular to the extracellular activity considering total volume of viable cells; CFU = colony forming units.].

Fig. 2

[⁶⁸Ga]Ga-DFO-B uptake in bacterial and fungal cells. (A) Bacterial and yeast cells were incubated with [⁶⁸Ga]Ga-DFO-B and [⁶⁸Ga]Ga-FCH for 45 min at 37°C with shaking under iron-depleted conditions. Uptake was expressed as ‘% AD/10⁹ CFU/mL’ and showed higher uptake of [⁶⁸Ga]Ga-DFO-B by S. aureus compared to other bacteria and yeast. (B) Uptake of [⁶⁸Ga]Ga-DFO-B by A. fumigatus in a 96-well filter plate comparing with other ⁶⁸Ga-siderophores. Uptake was performed by incubating 180 µL of mycelial cells with [⁶⁸Ga]Ga-DFO-B for 45 min at 37°C with shaking under iron-depleted conditions and expressed as ‘% AD/180 µL mycelial cells. Results showed poor uptake of [⁶⁸Ga]Ga-DFO-B by A. fumigatus. Error bars indicate standard deviations from three individual samples. [% AD: ‘% Added Dose’].