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Immunology and Infection

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Published: June 14th, 2021



1Laboratory of Ion Channel Research (LICR), VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium & Department of Cellular and Molecular Medicine, KU Leuven, 2Biomedical MRI, Department of Imaging & Pathology, KU Leuven, 3Belgium & KU Leuven Centre of Microbial and Plant Genetics, VIB-KU Leuven Center for Microbiology, Leuven, 4Laboratory of Experimental Urology, Department of Development and Regeneration, KU Leuven

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.

For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.

This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

Urinary tract infections (UTI) are among the most common bacterial infections in humans. Almost half of all women will experience a symptomatic UTI during their lifetime1. Infections limited to the bladder can give rise to urinary symptoms such as increase in urinary frequency, urgency, hematuria, incontinence, and pain. When the infection ascends to the upper urinary tract, patients develop pyelonephritis, with malaise, fever, chills, and back pain. Furthermore, up to 20% of patients with UTI suffer from recurrent infections resulting in a dramatic decrease in antibiotic sensitivity2,3....

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All animal experiments were conducted in accordance with the European Union Community Council guidelines and were approved by the Animal Ethics Committee of KU Leuven (P158/2018).

1. Culturing bacteria (adapted from7,13,14)

  1. Preparation
    1. Choose a luminescent UPEC strain that best fits the experimental needs.
      ​NOTE: Here, the clinical cystitis isolate, UTI89 (E. .......

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In vivo BLI correlates with CFU of the inoculum at time of instillation.
To evaluate the detection limit of BLI in vivo and the correlation with CFU of the inoculum, mice were infected with different concentrations of UTI89-lux and PBS as a negative control. Before instillation, uninfected animals were scanned to determine the background luminescence. Subsequent images were obtained immediately post-instillation (Figure 1A). .......

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Advantages of BLI compared to CFU counts
Longitudinal data
A major disadvantage of the traditional method of counting CFU to quantify microbial burden is the requirement of post-mortem organ homogenates, providing only one cross-sectional data point per animal. Conversely, BLI enables non-invasive longitudinal follow-up of infected animals. The animals can be imaged 2 to 3 times a day, providing detailed insight into the kinetics of the infection. Additionally, repeated measures of t.......

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This work was supported by grants from the Research Foundation - Flanders (FWO Vlaanderen; G0A6113N), the Research Council of KU Leuven (C1-TRPLe; T.V. and W.E.) and the VIB (to T.V.). W.E. is a senior clinical researcher of the Research Foundation - Flanders (FWO Vlaanderen). The strain UTI89-lux was a generous gift from Prof. Seed's laboratory13.


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Name Company Catalog Number Comments
96-well Black Flat Bottom Polystyrene Plate Corning 3925 for in vitro imaging
Aesculap ISIS Aesculap GT421 hair trimmer, with GT608 cap
Anesthesia vaporizer Harvard apparatus limited N/A
Baytril 100 mg/mL Bayer N/A Enrofloxacin
BD Insyte Autoguard 24 GA BD 382912 Yellow angiocatheter, use sterile plastic tip for instillation
C57Bl/6J mice Janvier N/A
Centrifuge 5804R Eppendorf EP022628146
Dropsense 16 Unchained Labs Trinean to measure OD 600nm
Dulbecco's Phosphate Buffered Saline, Gibco ThermoFisher Scientific REF 14040-083
Ethanol 70% denaturated 5L VWR international 85825360
Falcon 14ml Round Bottom Polystyrene Tube, Snap-Cap Corning 352057
Falcon 50ml cellstart Greiner 227285
Hamilton GASTIGHT syringe, PTFE luer lock, 100 µL Sigma-Aldrich 26203 to ensure slow bacterial instillation of 50 µL
Inoculation loop Roth 6174.1 holder: Art. No. 6189.1
Iso-Vet 1000mg/g Dechra Veterinary products N/A Isoflurane
IVIS Spectrum In Vivo Imaging System PerkinElmer REF 124262 imaging device
Kanamycine solution 50 mg/mL Sigma-Aldrich CAS 25389-94-0
Living Imaging Software PerkinElmer N/A BLI acquisition software, version 4.7.3
Luria Bertani Broth Sigma-Aldrich REF L3022 alternatively can be made
Luria Bertani Broth with agar Sigma-Aldrich REF L2897 alternatively can be made
Petri dish Sterilin 90mm ThermoFisher Scientific 101VR20 to fill with LB agar supplemented with Km
Pyrex Culture flask 250 mL Sigma-Aldrich SLW1141/08-20EA
Slide 200 Trinean Unchained Labs 701-2007 to measure OD 600nm
UTI89-lux N/A N/A Generous gift from Prof. Seed
Vortex VWR international 444-1372

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