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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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. .......

Representative Results

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). .......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
96-well Black Flat Bottom Polystyrene PlateCorning3925for in vitro imaging
Aesculap ISISAesculapGT421hair trimmer, with GT608 cap
Anesthesia vaporizerHarvard apparatus limitedN/Ahttps://www.harvardapparatus.com/harvard-apparatus-anesthetic-vaporizers.html
Baytril 100 mg/mLBayerN/AEnrofloxacin
BD Insyte Autoguard 24 GABD382912Yellow angiocatheter, use sterile plastic tip for instillation
C57Bl/6J miceJanvierN/A
Centrifuge 5804REppendorfEP022628146
Dropsense 16Unchained LabsTrineanto measure OD 600nm
Dulbecco's Phosphate Buffered Saline, GibcoThermoFisher ScientificREF 14040-083
Ethanol 70% denaturated 5LVWR international85825360
Falcon 14ml Round Bottom Polystyrene Tube, Snap-CapCorning352057
Falcon 50ml cellstartGreiner227285
Hamilton GASTIGHT syringe, PTFE luer lock, 100 µLSigma-Aldrich26203to ensure slow bacterial instillation of 50 µL
Inoculation loopRoth6174.1holder: Art. No. 6189.1
Iso-Vet 1000mg/gDechra Veterinary productsN/AIsoflurane
IVIS Spectrum In Vivo Imaging SystemPerkinElmerREF 124262imaging device
Kanamycine solution 50 mg/mLSigma-AldrichCAS 25389-94-0
Living Imaging SoftwarePerkinElmerN/ABLI acquisition software, version 4.7.3
Luria Bertani BrothSigma-AldrichREF L3022alternatively can be made
Luria Bertani Broth with agarSigma-AldrichREF L2897alternatively can be made
Petri dish Sterilin 90mmThermoFisher Scientific101VR20to fill with LB agar supplemented with Km
Pyrex Culture flask 250 mLSigma-AldrichSLW1141/08-20EA
Slide 200 TrineanUnchained Labs701-2007to measure OD 600nm
UTI89-luxN/AN/AGenerous gift from Prof. Seed
VortexVWR international444-1372

References

  1. Foxman, B. Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. American Journal of Medicine. 113 (1), 5-13 (2002).
  2. O'Brien, V. P., Hannan, T. J., Nielsen, H. V., Hultgren, S. J.

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Bioluminescence ImagingUrinary Tract InfectionsUTILongitudinal Follow upBacterial CultureInoculum PreparationAnesthesiaIntravesical Instillation

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