Multimodal Tracking of Controlled Staphylococcus aureus Infections in

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Multimodal tracking of (controlled) Staphylococcus aureus infections in mice Mick M. Welling, Clarize M de Korne, Silvia spa, Danny M. van Willigen, Albertus W Hensbergen, Anton Bunschoten, Nikolas Duszenko, Wiep Klaas Smits, Meta Roestenberg, and Fijs W.B. van Leeuwen ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00015 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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ACS Infectious Diseases

Multimodal tracking of (controlled) Staphylococcus aureus infections in mice Mick M. Welling a#, Clarize M. de Korne a,c, Silvia. J. Spa a, Danny M. van Willigen a, Albertus W. Hensbergen a, Anton Bunschoten

a,b,

Nikolas Duszenko

a,c,

Wiep Klaas Smits d, Meta

Roestenberg c, Fijs W. B. van Leeuwen a, b

Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, Leiden, the Netherlands a

Laboratory of BioNanoTechnology, Department of Agrotechnology and Food Sciences, Wageningen University & Research, Wageningen, the Netherlands b

Department of Parasitology and Department of Infectious Diseases, Leiden University Medical Center, Leiden, the Netherlands c

Department of Medical Microbiology, Section Experimental Bacteriology, Leiden University Medical Center, Leiden, the Netherlands d

# Corresponding author; Interventional Molecular Imaging Laboratory, Department of Radiology, S2-S Leiden University Medical Center Albinusdreef 2 2333ZA, Leiden, the Netherlands E-mail: [email protected] Phone: 0031-71-5262042

Keywords: Bacterial Infection, cell-tracking, Ubiquicidin, multimodal, SPECT, Fluorescence.

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There is a need to develop diagnostic and analytical tools that allow non-invasive monitoring of bacterial growth/dissemination in vivo. For such cell-tracking studies to hold translational value to controlled human infections, in which volunteers are experimentally colonized, they should not require genetic modification and allow tracking over a number of replication cycles. To gauge if an antimicrobial peptide tracer 99mTc-UBI29−41-Cy5, which contains both a fluorescent and radioactive moiety, could be used for such in vivo bacterial tracking, we performed longitudinal imaging of a thigh muscle infection with 99mTc-UBI29−41-Cy5 labelled Staphylococcus aureus. Mice were imaged using SPECT and fluorescence imaging modalities at various intervals during a 28 h period. Biodistribution analyses were performed to quantitate radioactivity in the abscess and other tissues. SPECT and fluorescence imaging in mice showed clear retention of the 99mTc-UBI29−41-Cy5-labelled bacteria following inoculation in the thigh muscle. Despite bacterial replication, the signal intensity in the abscess only modestly decreased within a 28 h period (52%ID/g at 4 h p.i. vs. 44%ID/g at 28 h p.i. (15% decrease)). After inoculation, a portion of the bacteria disseminated from the abscess and S. aureus cultures were obtained from radioactive urine samples. Bacterial staining with 99mTcUBI29−41-Cy5 allowed non-invasive bacterial cell tracking during a 28 h period. Given the versatility of the presented bacterial tracking method, we believe that this concept could pave the way for precise imaging capabilities during controlled human infection studies.


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To contribute to the development of innovative antimicrobial drugs and vaccines, controlled human infection (CHI) trials are being initiated to find solutions for monitoring bacterial persistence and better understanding the immune parameters involved.1, 2 In such studies, volunteers are purposefully colonized with e.g. bacteria in order to gain mechanistic insight into the potential of these pathogens to colonize tissue, and to study the interaction of specific bacteria with the existing microbiome and the native immune system. These studies have led to remarkable breakthroughs with regards to understanding local microbiome interaction (for example, experimental Neisseria lactamica colonisation in healthy students protected from Neisseria meningitis carriage) or interactions between different pathogens (live attenuated influenza vaccines increase pneumococcal carriage likelihood).

3, 4

Longitudinal molecular imaging of these experimental bacterial infections could help provide better insights into the dynamics of colonization and metastatic abscess formation and allow monitoring during antimicrobial interventions in animal models and potentially in future CHIs. Such investigation of bacterial colonization in humans would benefit from methods for tracking bacteria using non-invasive imaging modalities.5 Thus far, dedicated imaging of bacterial infections in pre-clinical studies mostly relies on optical imaging of genetically modified bioluminescent strains that express plasmid-integrated luciferase genes,6 or ultrasound imaging of strains that express acoustic reporter genes.7 While a useful pre-clinical tool, the use of genetically modified organisms (GMO’s) purely for clinical imaging purposes is not considered to be acceptable for safety reasons. An alternative approach is using molecular imaging techniques8,

9

for labelling of bacteria employing tracers with

fluorescent,10-12 magnetic,13 or radioactive imaging labels.14 In these cases the ability to label bacteria with tracers provides a non-GMO approach for tracking bacteria in pre-clinical models after in vitro labelling of bacteria. 3 ACS Paragon Plus Environment


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In this study, we used Staphylococcus aureus as a model system for studying experimental bacterial colonization, as these bacteria are well known for their potential to disseminate and cause metastatic abscesses.15, 16 The UBI29-41 peptide was selected because of its proven ability to label bacteria in vitro and in vivo.10, 17, 18To support longitudinal in vivo cell tracking during a 28 h period, we labelled the bacteria with a derivative of the previously reported hybrid ubiquicidin peptide 99mTc-UBI29-41-Cy5-N3, which provides a new addition to the hybrid-UBI familly.19 We assessed the effect of labelling on bacterial viability and the strength of both radioactive and fluorescent signals after various replication cycles in vitro. Subsequently, using our S. aureus model, we monitored the biodistribution and bacterial dissemination of labelling S. aureus after inoculation into a thigh muscle.


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RESULTS & DISCUSSION Both radioactive and fluorescent imaging can be used to monitor the progression of a Staphylococcus aureus thigh muscle infection in a mouse model using wild type bacteria. The strength of such a multimodal bacterial cell-tracking concept lies in the fact that establishment of infection and subsequent dissemination of bacteria can be monitored in a non-invasive manner using the radioactive signature. Fluorescence imaging complements radioactive findings with cellular imaging resolution and optical inspection of infected sites.

Radiolabelling of S. aureus with UBI29-41-Cy5-N3 A new UBI analogue UBI29-41-Cy5-N3 was synthesized (see supporting information). Tracer stability was determined in PBS and FCS; after incubating for 20 h at 37 oC, less than 10% release of radioactivity was observed (Fig 1A). As shown in Fig 1B, the labeling of S. aureus with 99mTc-UBI29-41-Cy5-N3 was optimally achieved (85-90%) after 1 h of incubation, and over the course of 20 h

99mTc-UBI 29-41-Cy5-N3

did not reveal dissociation from the bacteria.

Fluorescence microscopy of a detailed section containing aggregated bacteria demonstrated that 99mTc-UBI29-41-Cy5-N3 accumulated in the bacterial membrane (Fig 2A & B).

Effect of bacterial replication on signal strength After labelling of the parental bacteria, their replication over time could cause dilution of the signal intensity. We tried to map the effect of such a dilution over a 28 h period; in vitro this is expected to present 4-5 bacterial replication cycles. After correcting for number of viable bacteria, we observed about 10 times reduction in both radioactivity and fluorescence signal strength per bacterium (Fig 3A & B). Remarkably, qualitative assessment via fluorescence 5 ACS Paragon Plus Environment


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microscopy at 28h revealed a clustering of Cy5 signal around aggregated bacteria, which could indicate there is a biased tracer allotment for the daughter cells remaining in the aggregates versus those daughter cells “budding off” (Fig 4).

SPECT imaging and biodistribution of

99mTc-UBI

29-41-Cy5-N3-labelled

bacteria in

mice Due to the 6 h half-life of 99mTc, the current set-up limited monitoring to the initial 28 h time span following infection. This time-span reflects approximately 5 cycles of bacterial replication20, 21 and a theoretical 32-fold dilution of signal/bacteria. SPECT imaging revealed a strong signal at the site of bacterial deposition (Fig 5A). Longitudinal biodistribution analysis yielded relatively constant focal radioactivity values of 51.9±19.5 %ID/g (4 h p.i.), 42.1±21.5 %ID/g (20 h p.i.), and 44.4±25.7 %ID/g (28 h p.i.) (Fig 5B & Table S1) and differences were not significant. Over time muscle-to-blood ratios increased significantly (p

Multimodal Tracking of Controlled Staphylococcus aureus Infections in

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