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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....
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)
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). .......
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.......
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.
....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 | https://www.harvardapparatus.com/harvard-apparatus-anesthetic-vaporizers.html |
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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