15th European Molecular Imaging Meeting
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Imaging Infection

Session chair: Wiktor Syymanski (Groningen, Netherlands); Catherine Chapon (Fontenay-aux-Roses, France)
 
Shortcut: PW22
Date: Friday, 28 August, 2020, 12:00 p.m. - 1:30 p.m.
Session type: Poster

Contents

Abstract/Video opens by clicking at the talk title.

421

Simple radiosynthesis of 68Ga-desferrioxamine B (68GaDFO) for infection imaging

Afnan Darwesh1, Rachel Wellman1, Vincenzo Abbate2, Margaret Cooper1, Mauricio Morais1, Samantha Terry1, Robert Hider2, Michelle Ma1, Philip Blower1

1 king's college london, Department of Imaging, Chemistry & Biology, London, United Kingdom
2 king's college london, Department of Analytical, Environmental & Forensic Sciences, London, United Kingdom

Introduction

Endovascular stent graft infections are difficult to diagnose.1 A PET radiotracer that is taken up selectively by microorganism could provide more specific detection than currently used [18F]FDG. The Fe3+ complex of DFO, a siderophore, is taken up by bacteria, making its isostructural 68Ga complex applicable for imaging infection.DFO is a clinically accepted drug,3 hence its 68Ga complex has a low regulatory barrier to clinical translation. Here, we report a GMP method for 68GaDFO preparation, its binding to Escherichia coli (E.coli) in vitro, and its in vivo pharmacokinetics in healthy mice

Methods

68GaDFO was radiolabelled by mixing DFO dissolved in water (20µl), aqueous solution of sodium acetate (3.6 M) (20/30 µl), and 68Ga chloride (200µl). The mixture was diluted with water to 1 ml then incubated for 10 min at room temperature. The radiochemical purity and chemical identity were assessed by TLC and HPLC. natGaDFO was added to 68GaDFO and analysed by LC-MS. Stability in human serum was measured by RP-HPLC after 60-min incubation at 37o C. 68GaDFO uptake in E. coli was studied in vitro and compared to 68Ga ferrichrome-C, Enterobactin, and siderophore-free 68Ga. Healthy Balb/C mice (n=3) were injected intravenously with 68GaDFO and PET/CT scanned dynamically for 60 min. Mice were sacrificed for organ harvesting at 60 min, and organs were counted in a gamma-counter

Results/Discussion

68GaDFO was labelled with high radiochemical purity (≥ 95%) as measured by TLC and HPLC.  68GaDFO demonstrated no binding to serum proteins at any time point by RP-HPLC. All 68Ga labelled siderophores showed higher E. coli uptake than siderophore free 68Ga, with 68Ga ferrichrome-C showing the highest uptake. 68GaDFO exhibited rapid renal excretion and low blood retention. Ex vivo biodistribution at 60 min showed most of the activity resided in urine

Conclusions

68GaDFO radiolabelling with GMP graded reagents gave a product with high radiochemical purity identified as a 1:1 complex of Ga3+ with DFO. The complex showed specific uptake in E. coli. 68GaDFO did not release 68Ga to serum proteins, indicating high stability of the compound in serum. 68GaDFO is rapidly cleared with no significant retention in any organ except kidneys. In vivo biodistribution of 68GaDFO on infected animal models is now planned

AcknowledgmentThis work was supported by Saudi Arabian Cultural Bureau in The UK
References
[1] Elieson, M, Mixton, T, Carpenter 2012,' Coronary Stent Infections',Case Reports, 39, 884–889, Texas.
[2] Rabsch, W,  Winkelmann, G 1991, 'The Specificity of Bacterial Siderophore Receptors Probed by Bioassays', Biol. Metals, 4, 244–250, Germany.
[3] Mobarra, N, Shanaki, M, Ehteram, H, Nasiri, H, Sahmani, M, Saedi, M, Goudarzi, M, Pourkarim, H, Azed, 2016, 'A Review on Iron Chelators in Treatment of Iron Overload Syndroms', Int. J. Hematol. stem cell Res.,10, 239–247, Iran.
Dynamic PET/CT images of 68Ga DFO
Dynamic PET/CT MIP images at 2 min (A) and 60 min (B) of 68Ga DFO after intravenous injection. 
Keywords: 68GaDFO, siderophore, desferrioxamine B, infection imaging
422

In vivo imaging of infection and immunity in nonhuman primate models of infectious diseases

Catherine Chapon2, Nidhal Kahlaoui2, Sophie Luccantoni2, Céline Mayet2, Thibaut Naninck2, Quentin Pascal2, Sabine Tricot2, Roger Le Grand1

1 CEA, DRF/Jacob/ImVA U1184/IDMIT, Fontenay-aux-Roses, France
2 CEA, DRF/Jacob/ImVA U1184/IDMIT/L3i, Fontenay-aux-Roses, France

Introduction

Although non-human primates (NHP) provide a good model for infectious diseases, their exploration for viral transmission and dissemination by in vivo imaging has not been used extensively. This can be explained by the limited access to adequate structures for imaging these large animals with a high resolution while having specific regulations according to the level of confinement for the human pathogens. Our main objectives are to develop minimally invasive technologies for the longitudinal monitoring of infections, host response and treatments in NHP.

Methods

NHP models for immune related disorders and human infectious diseases (SIV, yellow fever, whooping cough, etc) for preclinical evaluation of human vaccines, immunotherapies and anti-microbial treatments are developed. In vivo imaging (near infrared fluorescence, probe based confocal endomicroscopy, echography, two-photon microscopy and PET-CT) is performed in BSL2 and BSL3 conditions. The two-photon microscope and the PET-CT camera suites are separated into two sides: a biologically “hot” side (pathogens present) and a biologically “cold” side (pathogens not present) (fig1). The imaging machines themselves are located outside of the containment on the cold side of the barrier wall and are thus accessible for adjustments and maintenance without requiring technicians to enter the hot side.

Results/Discussion

The behavior of skin antigen presenting cells following intradermal immunization with different vaccine vectors was characterized using probe based confocal endomicroscopy in order to better understand the mechanisms leading to the induction of cellular and humoral immune responses after vaccination1,2. Furthermore, non-invasive in vivo imaging procedures were developed to track bacterial localization and cellular interactions with host cells in the lower respiratory tract of challenged and naturally infected animals in a model of whooping cough in baboons3. Development of PET-CT approaches using immuno-PET or PET reporter gene imaging to track pathogens and immune cells at the whole body scale is ongoing.

Conclusions

This type of approaches could now be used for extended characterization at whole body and cellular resolution of diverse cell types in vivo and their interactions with other vaccine antigens and/or pathogens.

References
[1] Todorova, B., Adam, L., Culina, S., Boisgard, R., Martinon, F., Cosma, A. et al. Electroporation as a vaccine delivery system and a natural adjuvant to intradermal administration of plasmid DNA in macaques. Sci Rep. 7, 4122  (2017)
[2] Todorova, B., Salabert, N., Tricot, S., Boisgard, R., Rathaux, M., Le Grand, R. et al. Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques. Contrast Media Mol Imaging. 2017, 3127908  (2017)
[3] Naninck, T., Coutte, L., Mayet, C., Contreras, V., Locht, C., Le Grand, R. et al. In vivo imaging of bacterial colonization of the lower respiratory tract in a baboon model of Bordetella pertussis infection and transmission. Sci Rep. 8, 12297  (2018)
PET-CT imaging facilities in BSL2&3 environment

Schematic of the PET-CT imaging facilities (Vereos®, Philips) with the hot side (BSL2&3) imaging room (in red) including the patient table, the containment barrier wall in the background with the circular containment tube extending into the cold side (in blue)

Keywords: infectious diseases, nonhuman primate, PET-CT, in vivo two-photon, in vivo confocal
423

Cellular and molecular mechanisms controllingintracellular GBS elimination in neonatal and adult macrophages and the role of Akt1 kinase

Ourania Kolliniati1, 2, Ioanna Lapi1, 2, Ioanna Pantazi1, 2, Eleni Vergadi1, 2, Christos Tsatsanis1, 2

1 University of Crete, School of Medicine, Heraklion, Greece
2 FORTH-Foundation for Research and Technology - Hellas, IMBB-Institute of Molecular Biology and Biotechnology, Heraklion, Greece

Introduction

Streptococcus Group Beta (GBS) is a commensal for healthy adults but a life threatening pathοgen for infants as it is the leading cause of sepsis and meningitis1.  Newborns depend on macrophage and neutrophil activity to eliminate bacteria2. Previous studies from our group showed that Akt1 kinase deletion polarizes macrophages towards M1 phenotype, resulting in increased oxidative burst, pro-inflammatory cytokines and enhanced bactericidal capacity3. Our aim is to study the defective mechanisms of GBS elimination in neonatal macrophages and determine whether Akt1 ablation can reinforce them.

Methods

Wild-Type (WT) adult and WT and Akt1LoxP/LoxP,LysMCre neonatal peritoneal macrophages were infected with a hyper-virulent strain of GBS (serotype III) for 2 hours to allow phagocytosis of bacteria.
Intracellular bacterial load was determined by serially diluting and plating macrophage lysates on Todd Hewitt Broth Agar plates.
For knock down, siRNAs were used for the gene of interest and their efficiency was quantified by real time qPCR.
Total RNA was isolated using the Trizol protocol and cDNA was synthesized. The relative mRNA levels of genes were measured by RT-q-PCR using RSP9 as a housekeeping gene.
Confocal microscopy was used to determine co-localization of GBS with autophagy-regulating proteins and Electron microscopy to visualize its subcellular localization.

Results/Discussion

Our data demonstrated that upon GBS infection Wild Type (WT) neonatal macrophages had more bacteria which also proliferated in the cytoplasm compares to neonatal Akt1-/- or WT adult macrophages, which appeared to eliminate GBS. Upon infection, WT neonatal cells produced more of the anti-inflammatory cytokine IL-10, known to limit immune responses, while all cells exhibited increased expression of Nox2, known to facilitate ROS generation within phagosomes.
To determine the contribution of autophagy in GBS elimination we knocked down Rubicon and Atg5 and showed that the intracellular GBS load was only increased in WT adult and Akt1-/- neonatal cells. LAMP-1 and LC3-II co-localized with GBS in WT adult and Akt1-/- neonatal macrophages but not WT neonatal cells while Transmition Electron microscopy showed that GBS resides in phagosomes and not autophagosomes. These data suggest that elimination of GBS occurs via LC3 Associated Phagocytosis (LAP), which is defective in WT neonates.

Conclusions

Overall, our work revealed the molecular mechanisms controlling GBS infection in adult and neonatal macrophages. We demonstrated that this process is tightly regulated by an alternative autophagy pathway known as LC3 Associated Phagocytosis (LAP) and ROS production, a process controlled by Akt1 kinase. Our findings propose a potential therapeutic role of Akt1 inhibition against neonatal GBS-borne diseases.

AcknowledgmentOur work has been funded by European Society of Pediatric Infectious Diseases (ESPID)  State Scholarships Foundation (IKY) and Hellenic Foundation for Research and Innovation (HFRI). Our research has been  further supported by funding received from the Bio-based Industries Joint Undertaking under the European Union Horizon 2020 research and innovation program under grant agreement No 790956  (AQUABIOPRO-FIT)
References
[1] Ouchenir, L., Renaud, C., Khan, S., Bitnun, A., Boisvert, A.-A., Mcdonald, J., … Robinson, J. L. (2017). The Epidemiology, Management, and Outcomes of Bacterial Meningitis in Infants. Pediatrics140(1). doi: 10.1542/peds.2017-0476
[2] Henneke, P., & Berner, R. (2006). Interaction of Neonatal Phagocytes with Group B Streptococcus: Recognition and Response. Infection and Immunity74(6), 3085–3095. doi: 10.1128/iai.01551-05 
[3] Androulidaki, A., Iliopoulos, D., Arranz, A., Doxaki, C., Schworer, S., Zacharioudaki, V., … Tsatsanis, C. (2009). The Kinase Akt1 Controls Macrophage Response to Lipopolysaccharide by Regulating MicroRNAs. Immunity31(2), 220–231. doi: 10.1016/j.immuni.2009.06.024
Keywords: LC3 Associated Phagocytosis (LAP), Group Beta Streptococcus (GBS), Akt1 kinase
424

Dose dependent luciferin boosts increase in vivo bioluminescence detection sensitivity at the early-onset of invasive aspergillosis in a murine infection model

Agustin Resendiz Sharpe1, Matthias Brock2, Paul E. Verweij3, Lore Vanderbeke1, Johan Maertens4, 1, Katrien Lagrou5, 1, Greetje Vande Velde6

1 KU Leuven, Department of Microbiology, Immunology and Transplantation, Leuven, Belgium
2 University of Nottingham, Fungal Biology Group, School of Life Sciences, Nottingham, United Kingdom
3 Radboud University Medical Center, Department of Medical Microbiology, Nijmegen, Netherlands
4 University Hospitals Leuven, Department of Hematology, Leuven, Belgium
5 University Hospitals Leuven, Department of Laboratory Medicine and National Reference Center for Mycosis, Leuven, Belgium
6 Biomedical MRI Unit/MoSAIC, Department of Imaging and Pathology, Leuven, Belgium

Introduction

Bioluminescence (BL) imaging has been successfully used to non-invasively monitor and quantify in vivo fungal infections in individual animals. However, in vivo BL imaging of fungi is challenged by lower BL signals compared to mammalian cells, probably due to cell-wall restricted uptake of luciferin. This hampers detection of the earliest stages of infection. Here, we explored the effect of increasing the in vivo luciferin dose on the sensitivity of detection of the early onset of pulmonary aspergillosis in a murine infection model.

Methods

Male BALB/c mice (10-weeks) were immunosuppressed with cyclophosphamide on day 4 and 1 prior to infection and day 2 post-infection (150  mg/kg). Animals were inoculated orotracheally with a suspension of 5  ×  105 conidia of an A. fumigatus strain expressing a red-shifted codon-optimized firefly luciferase. Luciferin-EF (Promega) was administered intraperitoneally at three different doses (126 mg/kg, 250 mg/kg, 500 mg/kg, n = 5 per group). Bioluminescence peak signal intensities (total photon flux) were acquired daily from day 1 to day 4 post-infection (IVIS SPECTRUM, Living image software; PerkinElmer) and quantified from a region of interest covering the lungs.

Results/Discussion

Overall, increasing luciferin from 126 mg/kg to 250 mg/kg elevated the photon flux by approximately 1- log or a 1.5 fold increase (p = <0.0001). A further doubling of the dose to 500 mg/kg increased the photon flux by another log or 4.5 fold increase (p=<0.0001) (Figure 1). At doses of 250 mg/kg and 500 mg/kg (p=<0.00001) invasive pulmonary aspergillosis was already detected at day 1 by significant total flux differences compared to baseline. In contrast, at 126 mg/kg detection was delayed to day 4 (p=0.032).
Boosting of BL signals was confirmed by topping up luciferin dose to a total concentration of 500 mg/kg in both the 126 mg/kg and 250 mg/kg luciferin dose groups on day 4 (Figure 2). This resulted in a significantly increased total photon flux of almost 1-log or 2.2 fold increase in the 126 mg/kg group and 1-log-or 1.8 -fold increase in the 250 mg/kg group. No significant differences in weight, survival and fungal burden (CFU counts) were observed between groups.

Conclusions

This study indicates that in an in vivo invasive pulmonary aspergillosis mouse model a dose of 500 mg/kg luciferin results in significantly increased total photon fluxes compared to the commonly used dose of 126 mg/kg or 250 mg/kg. Moreover, boosting with luciferin significantly increased the sensitivity of detection of infection to day 1 post-inoculation compared to day 4 at the commonly used dose with no indications of increased toxicity.

References
[1] Aswendt M, Adamczak J, Couillard-Despres S, Hoehn M. Boosting Bioluminescence Neuroimaging: An Optimized Protocol for Brain Studies. PLoS One. 2013;8(2):55662. doi:10.1371/journal.pone.0055662
[2] Andreu N, Zelmer A, Fletcher T, et al. Optimisation of Bioluminescent Reporters for Use with Mycobacteria. Doherty TM, ed. PLoS One. 2010;5(5):e10777. doi:10.1371/journal.pone.0010777
[3] Brock M. Application of bioluminescence imaging for in vivo monitoring of fungal infections. Int J Microbiol. 2012. doi:10.1155/2012/956794
[4] Poelmans J, Himmelreich U, Vanherp L, et al. A multimodal imaging approach enables in vivo assessment of antifungal treatment in a mouse model of invasive pulmonary aspergillosis. Antimicrob Agents Chemother. 2018. doi:10.1128/AAC.00240-18
Figure 1. – Comparison of in vivo total flux according to luciferin dose in an invasive aspergillosi

The graph represents the total log10 photon flux as quantified from region of interest (complete lung region) of the BL images acquired from day 1 to day 4 with luciferin doses of 126 mg/kg, 250 mg/kg and 500 mg/kg. Error bars represent SD of the results from multiple mice (n = 5). Repeated measures ANOVA (p=0.0164) with multiple comparison analysis between doses (right) and to baseline (graph) of transformed data; p= 0.03 (*), 0.006 (**), 0.0006 (***), <0.0001 (****).

Figure 2. – Total photon flux from initial and topped-up luciferin doses in an in vivo invasive aspe
Graph representing total photon flux measurements at day 4 post infection with initial doses (126, 250 and 500 mg/kg; solid bars) and when topped up to 500 mg/kg (shaded bars). Error bars represent SD of the results from multiple mice (n = 5). (Repeated measures ANOVA with multiple comparison analysis, * p= 0.05)
Keywords: Fungal infections, in vivo bioluminescence, luciferin dose boost
425

Preparing optimal doses of 68Ga-labeled ubiquicidin-29-41 for efficient imaging of infection in a small patient population

Mariza Vorster1, Jan Rijn Zeevaart1, Mike M. Sathekge1, Thomas Ebenhan1

1 University of Pretoria, Nuclear Medicine, Pretoria, South Africa

Introduction

The ubiquicidin fragment 29-41 of (UBI) has recently been conjugated with radiometal chelators such as 1,4,7-triaza-cyclononane-1,4,7-triacetic-acid for prospective use as a tracer in nuclear medicine for imaging of infection (1,2). We developed radiolabeling solution for [68Ga]Ga-UBI followed by application in non-human primates (3). Herein, we investigate a kit-based tracer production towards good clinical practice including tracer administration, [68Ga]Ga-UBI uptake and excretion. This will allow for improved utilization of [68Ga]Ga-UBI-PET/CT imaging in infectious diseases.

Methods

Fifteen [68Ga]Ga-UBI (9.2±3.8 µM) productions were performed; radiolabeling was carried out as detailed (1). Ethical approval for tracer injections was given. Six male (19-57 y, 51-70 kg) and eight female (18-57 y, 18-110 kg) subjects with suspected infection received [68Ga]Ga-UBI followed whole body PET/CT imaging. Urinary radioactivity (kidneys/bladder) and organ uptake was expressed as percentage of the injected dose (%ID).

Results/Discussion

Radiolabeling yielded 473 ± 324 MBq of safe-to-inject [68Ga]Ga-UBI in 25 ± 11 min (%LE 51±23), %RCP >96.5. Occasional losses occurred due to colloidal-68Ga (-17±12%) and C18-cartridge retained activity (-19 ±18%). [68Ga]Ga-UBI formulations met the requirements for radiopharmaceutical production and were safely administered to patients (5.0±1.6 mCi) as follows: the average activity doses, molar activities and injected tracer masses were 3.0±1.2 MBq/kg; 25±12 MBq/nmol and 8.5±4.4 nmol, respectively. Normal biodistribution included excretion of 34-57 %ID in kidneys/bladder and < 2.8% ID in liver > heart > spleen > bone > muscle > 0.06 %ID. Clinical indications for imaging included suspected tuberculosis, musculoskeletal infections, cholecystitis, endocarditis and nephritis.

Conclusions

Our production of [68Ga]Ga-UBI met the quality requirements and release criteria for human use and offers a convenient diagnostic tool to study infetious diseases non-invasively. Our study represents one of the bigger patient cohorts with suspected infection imaged with [68Ga]Ga-UBI-PET/CT.

Acknowledgment

The authors thank the staff at the Department of Nuclear Medicine, University of Pretoria and the Department of Radioachemistry at the South African Nuclear Energy Corporation.

References
[1] Ebenhan, T, Zeevaart, JR, Venter, JD, Govender, T, Kruger, GH, Jarvis, NV and  Sathekge MM. 2014, Preclinical evaluation of 68Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid-ubiquicidin as a radioligand for PET infection imaging.’, J Nucl Med., 55(2), 308-314.
[2] Vilche, M, Reyes, AL, Vasilskis, E, Oliver, P, Balter, H and Engler, H. 2016 ‘⁶⁸Ga-NOTA-UBI-29-41 as a PET Tracer for Detection of Bacterial Infection.’ , J Nucl Med., 57(4), 622-627.
[3] Ebenhan, T, Sathekge, MM, Lengana, T, Koole, M, Gheysens, O, Govender, T and Zeevaart, JR. 2018 ’68Ga-NOTA-Functionalized Ubiquicidin: Cytotoxicity, Biodistribution, Radiation Dosimetry, and First-in-Human PET/CT Imaging of Infections.’, J Nucl Med., 59(2), 334-339.
[4] Bhusari, P, Bhatt, J, Sood, A, Kaur, R, Vatsa, R, Rastogi, A, Mukherjee, A, Dash, A, Mittal, BR and Shukla, J., 2019, ‘Evaluating the potential of kit-based 68Ga-ubiquicidin formulation in diagnosis of infection: a pilot study68Ga.’, Nucl Med Commun., 40(3), 228-234.
Keywords: [68Ga]-UBI, PET Infection imaging, kit-based infection imaging
426

Presenting a codon-optimized palette of fluorescent proteins for detection of Candida albicans under in vitro conditions and in animal infection models

Wouter Van Genechten1, 2, 3, Liesbeth Demuyser1, 2, Peter Dedecker3, Patrick Van Dijck1, 2

1 VIB-KU Leuven Center for Microbiology, VIB, Leuven, Belgium
2 KU Leuven, Biology, Laboratory of Molecular Cell Biology, Leuven, Belgium
3 KU Leuven, Chemistry,, Leuven, Belgium

Introduction

Fluorescent proteins with varying colors are indispensable tools for the life sciences research community. These fluorophores are often developed for use in mammalian systems. However, the successful application of these labels in other organisms in the tree of life, such as the fungus Candida albicans, can be difficult to achieve due to the difficulty in engineering constructs for good expression in these organisms. Especially for C. albicans this poses a problem as this species translates a CUG codon mostly into a serine instead of a leucine.

Methods

We explored a number of different in silico optimization tools (Integrated DNA technologies, OPTIMIZER, ATGme, manual adaptation) to generate a whole pallet of fluorescent proteins ranging from cyan to red and including photoswitchable fluorophores that can be used in combination with super resolution microscopy. Brightness assessment was determined using fluorescence activated cell sorting as well as fluorescence microscopy.

Results/Discussion

We show that none of the optimization strategies is generally applicable, and that even very closely related proteins require the application of different strategies to achieve good expression. We show that by using the codon-optimized photoswitchable fluorophores in combination with stochastic optical fluctuation imaging (PcSOFI) we can now visualize expression of genes that previously could only be visualized upon overexpression. We are also using the new fluorophores to visualize the specific morphology (yeast or hyphal cells) of C. albicans that we then will use in our different animal models, such as upon oral, vaginal or intestinal infection.

Conclusions

In addition to reporting new fluorescent protein variants for applications in Candida albicans, our work highlights the ongoing challenges in optimizing protein expression in heterologous systems. The tools we are currently developing will be important to visualize endogenous expression of genes both under in vitro as well as in animal model experiments.

Acknowledgment

This work was supported by the Research Foundation Flanders (FWO Vlaanderen) [1S01817N to W.V.G., G062616N to P.V.D. and P.D.]. We thank Celia Lobo Romero for the generous help in creation of the strains, Dr. Siewert Hugelier for statistical insights and Prof. Johan Robben for the fruitful discussion on codon optimization.

References
[1] Moeyaert, B, Dedecker, P, 2014, 'PcSOFI as a smart label-based superresolution microscopy technique', Methods Mol Biol, 1148, 261-276, Springer.
Keywords: Candida albicans, fluorescent proteins, fungal infection model, super-resolution microscopy
428

Screening of gallium-68 labelled siderophores for developing PET-imaging agents as a non-invasive diagnostic tool

Asma Akter1, 2, Ruslan Cusnir2, 3, Michelle Ma2, Margaret Cooper2, Amita Patel4, Samantha Terry2, Nicholas Price4, Oliver Lyons5, Kenneth Bruce6, Robert Hider7, Philip Blower2, Vincenzo Abbate1

1 King's College London, Department of Analytical, Environmental and Forensic Sciences, London, United Kingdom
2 King’s College London, St Thomas’ Hospital, School of Biomedical Engineering and Imaging Sciences, London, United Kingdom
3 Paul Scherrer Institute, Laboratory of Radiochemistry, Villigen, Switzerland
4 King's College London and Guy's and St Thomas' NHS Foundation Trust, Centre for Clinical Infection and Diagnostics Research, Department of Infectious Diseases,, London, United Kingdom
5 Guy’s & St Thomas’ NHS Foundation Trust, Academic Department of Surgery, Cardiovascular Division, London, United Kingdom
6 King's College London, Molecular Microbiology Research Laboratory, Pharmaceutical Science Research Division, London, United Kingdom
7 King's College London, Institute of Pharmaceutical Science, London, United Kingdom

Introduction

The prevalence of microbial vascular graft infections (VGIs) is estimated between 0.5–6% 1. Current invasive removal of the infected prosthesis and prolonged antimicrobial therapy2 demand a single, sensitive and specific diagnostic test. Recent advances in radiolabelled siderophores3, exploring siderophore-mediated iron transport, have inspired PET-imaging of microbial infections4. Chemical similarity of gallium to iron might therefore permit non-invasive diagnosis of VGIs by PET imaging with 68Ga and possibly systemic targeted radionuclide therapy with the Auger electron emitter 67Ga.

Methods

Selected siderophores (enterobactin (ENT), deferoxamine (DFO), ferrichrome C (FC), pyoverdine (PVD) and schizokinen (SKN)) were labelled with 68Ga in acetate buffer. Quality control was performed by iTLC. In vitro 68Ga-siderophore uptake was performed with Escherichia coli and Pseudomonas aeruginosa in different media under iron-depleted and iron-supplemented (10 µM FeCl3) conditions. Iron-siderophore complexes were used as uptake blocking agents. The results were expressed as intracellular to extracellular distribution concentration ratio of the radioactivity. In vitro uptake studies in stents were performed with 68Ga labelled FC/DFO, where stents were incubated with E. coli, Staphylococcus aureus, P. aeruginosa and Candida albicans; autoradiography and PET/CT images were taken.

Results/Discussion

Radiochemical purity of all the siderophores labelled with 68Ga was > 95%. Since the microbial load in stents is expected to be lower than other infections5, this study aims to assess a range of bacterial load expressed as CFU/mL. In vitro uptake studies in E. coli (109 CFU in 1 mL) with 68Ga-labelled ENT, FC, and SKN showed high intracellular uptake (distribution ratio) of the radioactivity, particularly in iron-depleted conditions and using stationary phase bacterial culture. Stationary phase P. aeruginosa (108 CFU in 1 mL)  showed specific uptake of DFO, FC, and PVD in low iron conditions. Uptake was blocked either by Ferri-ENT/ Ferri-PVD complex (>15 µM). In vitro uptake studies in stents with 68Ga-DFO showed uptake in S. aureus and P. aeruginosa but not in E. coli and C. albicans. Stent experiments with 68Ga-FC in E. coli showed uptake which was visualized with autoradiography and PET/CT imaging.

Conclusions

Our studies show in vitro uptake of selected siderophores in Gram-negative bacteria and give the basis for further testing in Gram-positive bacteria, associated with VGIs. Additionally, the uptake of 68Ga-DFO/FC by P. aeruginosa, S. aureus and E. coli in stents points the way to developing a non-invasive diagnostic for VGIs.

Acknowledgment

The work is funded by King’s Health Partner’s Research and Development Challenge Fund, 2019.

References
[1] Ajdler-Schaeffler E, Scherrer A, Keller PM, Zbinden R, Anagnostopoulos A, Hofmann M, Rancic Z, Zinkernagel AS, Bloemberg GV, Hasse BK. Increased pathogen identification in vascular graft infections by the combined use of tissue cultures and 16S rRNA Gene Polymerase chain reaction. Front Med. 2018; 5:169.
[2] Lyons OT, Baguneid M, Barwick TD, Bell RE, Foster N, Homer-Vanniasinkam S, Hopkins S, Hussain A, Katsanos K, Modarai B, Sandoe JA. Diagnosis of aortic graft infection: a case definition by the Management of Aortic Graft Infection Collaboration (MAGIC). Eur J Vas and Endo Sur. 2016;52(6):758-63.
[3] Hider RC, Kong X. Chemistry and biology of siderophores. Nat Prod Rep. 2010; 27:637-657.
[4] Petrik M, Zhai C, Haas H, Decristoforo C. Siderophores for molecular imaging applications. Clin Transl Imaging. 2017;5(1):15-27.
[5] Herten M, Bisdas T, Knaack D, Becker K, Osada N, Torsello GB, Idelevich EA. Rapid in vitro quantification of S. aureus biofilms on vascular graft surfaces. Front Microbiol. 2017; 8:2333.
In vitro uptake of 68Ga-siderophores in bacteria

Figure 1: (a) ENT, SKN and FC labelled with 68Ga uptake by Mid-log phase E. coli cells with 109 CFU/mL. The uptake was performed in Minimal Medium 9 (MM9). Ferri-ENT (>15 µM) was used as an uptake blocking agent. (b) PVD, DFO and FC labelled with 68Ga uptake by stationary phase P. aeruginosa cells with 108 CFU/mL. The uptake was performed in Succinate medium (SM). Ferri-PVD (> 15 µM) was used as an uptake blocking agent. The uptake was expressed as intracellular to extracellular distribution ratio of the radioactivity. Biological triplicates were performed.

In vitro uptake in stents

Figure 2: PET/CT image of two sterile and four infected pieces of vascular stent after incubated with 68Ga-ferrichrome C (top), further imaging with autoradiography (middle) and before imaging (bottom).

Keywords: 68Ga, Stent graft infections, Siderophores, Imaging, in vitro
430

In vitro evaluation of 68Ga-DOTA-CDP1 in selected bacteria towards imaging infection with PET

Amanda H. Mdlophane1, 2, 3, Thomas Ebenhan1, 3, Sanah Nkadimeng4, Mike M. Sathekge1, 3, Jan Rijn Zeevaart2, 3, 5

1 University of Pretoria & Steve Biko Academic Hospital, Nuclear Medicine, Pretoria, South Africa
2 The South African Nuclear Energy Corporation (Necsa), Radiochemistry, Brits, South Africa
3 Nuclear Medicine Research Infrastructure (NuMeRI), Preclinical Imaging Facility, Brits, South Africa
4 University of Pretoria, Phytomedicine Proramme, Department of Paraclinical Sciences, Faculty of Veterinary Science, Onderstepoort, South Africa
5 Department of Science and Technology, Preclinical Drug Development Platform, North West University, Potchefstroom, South Africa

Introduction

68Ga-DOTA-CDP1 was recently radiosnythesised due to its antimicrobial properties for its potential as a nuclear medicine diagnostic tracer for complicated infections.1 The aim was to ascertain the binding of 68Ga- DOTA-CDP1 onto the bacterial cell envelope and to note any morphological changes as a result of exposure to various peptide concentrations.

Methods

Bacterial cell binding of 68Ga-DOTA-CDP1 with/without pre-treatment with DOTA-CDP1 was determined in the following microbes: Escherichia coli (EC), Staphylococcus aureus (SA) and Mycobacterium smegmatis (MS). Treatment groups included 68Ga-DOTA-CDP1 (0.33 µg/ml) with rising concentrations of DOTA-CDP1 [5 µg (1.7µg/ml), 10 µg (3.3µg/ml), 25 µg (8.3µg/ml) and 50 µg (16.7 µg/ml)] per 1.5 x108 CFU and the untreated group contained only 68Ga-DOTA-CDP1. 68GaCl3 and 68Ga-NODAGA were used as positive and negative controls. Bacterial cell envelope morphological changes, as a result of the treatments, were analysed through transmission electron microscopy (TEM).

Results/Discussion

MS: 68Ga-DOTA-CDP1 uptake ranged from 16–56%. MS50µg and MS25µg treatment had higher uptake than all at both time points. Tracer uptake was significantly higher than 68Ga-NODAGA (p<0.0004) with no difference compared to 68GaCl3. SA: Accumulation ranged from 22–46% and higher uptake with SA50µg (46±5.0%) than the untreated group (SA0: 22.8±5.0%, p<0.04) after 1 h. EC: Uptake ranged from 25-45%. Untreated bacteria (EC0) had lower uptake than EC25µg (40 ± 1.6%; p<0.01), EC10µg (42±0.4%; p<0.02), and EC5µg (44±0.9%; p<0.01) treatment groups. At 0 h, uptake was notably higher (p<0.02) in MS50µg (56±3.0%) contrary to EC50µg (41±2.5 %) and SA50µg (41±1.6%, p<0.01). Untreated MS0 was also lower than untreated EC0 at both times (0 h: p<0.01; 1 h: p<0.02).TEM showed leakage of cytoplsm, ghost cells and cell wall detachment with high DOTA-CDP1 concentrations in EC and SA. This may interfere with detection due to disintergrated bacteria. MS morphology remained unchanged.

Conclusions

68Ga-DOTA-CDP1, binds to the cell envelope without damaging it, however, concentration dependent accumulation in the presence of excess DOTA-CDP1 led to the destruction of the cell wall and leakage of cytoplasmic contents which is congruent with induced cell lysis and cell death. Thus, 68Ga-DOTA-CDP1 concentration must be kept at sub-therapeutic levels to enable imaging using PET.

Acknowledgment

This study supported the Nuclear Medicine Research Infrastructure (NuMeRi), a national technology platform developed and managed by the South African Nuclear Energy Corporation SOC Ltd (Necsa) and funded by the Department of Science and Technology

 

References
[1] Mdlophane, AH, Ebenhan, T 2019, ‘Comparison of DOAT and NODAGA chelates for 68Ga-labelled CDP1 as novel infection PET imaging agents’, J. Radioanal Nucl. Chem., 322, 629-638, Budapest: Akademiai Kiado
Keywords: 68Ga-DOTA-CDP1, infection imaging, PET