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&#39;s laboratory13.

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)
<ol>
	<li>Preparation
	<ol>
		<li>Choose a luminescent UPEC strain that best fits the experimental needs. 
		&#8203;NOTE: Here, the clinical cystitis isolate, UTI89 (E. coli), was selected because of its uropathogenic capacity in both humans and rodents, and its common use in murine UTI models5,7,15. The bioluminescent isogenic strain carrying the complete lux operon (luxCDABE) is further referred to as UTI89-lux. This operon also carries kanamycin sulfate (Km) resistance genes. Therefore, Km can be used to select bioluminescent colonies13.</li>
		<li>Make a correlation curve for the CFU and optical density at 600 nanometers (OD 600 nm) for the chosen bacterial strain7.
		<ol>
			<li>To do this, culture bacteria (using the protocol below) and make 8 different dilutions in phosphate buffered saline (PBS). Measure the OD at 600 nm for all these dilutions and plate them on Luria-Bertani (LB) plates to determine the CFU.</li>
			<li>Plot the OD 600 nm values and the CFU values to determine the correlation and to obtain a standard curve. 
			NOTE: For future experiments, measure the OD at 600 nm and use this standard curve to get an instant estimation of the CFU in a bacterial solution. 
			&#8203;CAUTION: UPEC are pathogenic bacteria, ensure rooms are properly equipped and use personal protective equipment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Three days before the instillation
	<ol>
		<li>Obtain single colonies by streaking out the glycerol stock of bacteria, using an inoculation loop, on LB plates supplemented with 50 &#181;g/mL Km and culture overnight at 37 &#176;C. Seal these plates with a paraffin film to store at 4 &#176;C up to 1 week.</li>
	</ol>
	</li>
	<li>Two days before the instillation
	<ol>
		<li>Fill a sterile 14 mL polystyrene round bottom tube with dual-position snap caps with 5 mL of LB broth supplemented with 50 &#181;g/mL Km. Pick a single bacterial colony with an inoculation loop and add this to the LB broth. Vortex for 10 s to ensure proper mixing.</li>
		<li>Culture statically with the snap cap in the open position at 37 &#176;C overnight. 
		&#8203;NOTE: Static growth of E. coli promotes the expression of type 1-pili, which are critical for the adherence to and invasion of urinary epithelial cells.</li>
		<li>Prepare a sterile Erlenmeyer or culture flask.</li>
	</ol>
	</li>
	<li>One day before the instillation
	<ol>
		<li>After the incubation, vortex the tube for 10 s to ensure proper homogenization of the bacterial culture. Make a sub-culture in the Erlenmeyer by adding 25 &#181;L of the bacterial suspension to 25 mL of fresh LB medium without antibiotics.</li>
		<li>Close the Erlenmeyer and culture statically at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>On the day of instillation
	<ol>
		<li>Vortex the Erlenmeyer for 10 s to ensure proper mixing.</li>
		<li>Pour the culture from the Erlenmeyer into a 50 mL culture tube and centrifuge at 3,000 x g and 4 &#176;C for 5 min. Decant the supernatant and resuspend the bacterial pellet in 10 mL of sterile PBS and vortex again for 10 s.</li>
		<li>Choose the experimental concentration of the inoculum (CFU) and use the standard curve obtained in step 1.1.2 to determine the corresponding OD 600 nm.</li>
		<li>Add 1 mL of the resuspended bacterial culture into 9 mL of sterile PBS and check the OD at 600 nm. Adjust further by adding either sterile PBS or concentrated bacterial solution, until the desired OD 600 nm is reached7. 
		NOTE: For example, for UTI89-lux adjust the culture by adding PBS until the OD 600 nm reaches 0.45, corresponding to 2 x 107 CFU/50 &#181;L. To verify the number of viable CFUs, make 10-fold serial dilutions and plate 50 &#181;L of the fifth and sixth 10-fold dilutions on LB plates in triplicate to obtain less than 300 colonies. Count the obtained colonies the next day after overnight incubation at 37 &#176;C. OD 600 nm gives an instant read-out but use CFU as quality control.</li>
	</ol>
	</li>
</ol>
2. Inoculation of the animals (adapted from7,16)
<ol>
	<li>Preparation of the animals
	<ol>
		<li>Choose the desired mouse strain(s) depending on the research question and availability of knock-out lines, experimental details, and differences in UTI susceptibility5,17. Keep in mind that transurethral catheterization is easier in female mice. Do not use animals younger than 8 weeks, as they are immunologically immature. Here, 12-week-old female C57Bl/6J mice were used.</li>
		<li>Order animals well in advance and let them acclimate, ideally for 7 days. Group house animals in individually ventilated cages, under standard 12 h light/dark conditions.</li>
		<li>Shave the abdominal region of the animals to limit the loss of signal. Do not use hair removal cream, as it can burn the skin of the animals quickly. Restrain the animal by tightly holding the scruff and hind limbs with the non-dominant hand while shaving with the dominant hand. Alternatively, shave under isoflurane anesthesia. 
		NOTE: Animals were shaved 2 days prior to imaging, considering the animals will groom the shaved area even further.</li>
		<li>Provide water and standard food ad libitum throughout the experiment. However, deprive animals from water 2 h prior to instillation to minimize the bladder volume during instillation.</li>
		<li>Mount a sterile 24 G angiocatheter tip on a 100 &#181;L syringe and fill the syringe with the bacterial solution prepared in step 1.5.3. 
		&#8203;NOTE: To determine the background luminescence, obtain a baseline image prior to the instillation (see step 3, below).</li>
	</ol>
	</li>
	<li>Instillation of the animals
	<ol>
		<li>Place the animals in an induction chamber and anesthetize them using inhalation of isoflurane with pure oxygen as a carrier gas (induction at 3% and maintenance at 1.5%).</li>
		<li>Place one animal on a working surface in the supine position and maintain a stable isoflurane anesthesia using a nose cone during the instillation. Apply the eye-ointment.</li>
		<li>Expel the residual urine by applying gentle compression and making circular movements on the suprapubic region. Clean the lower abdomen with 70% ethanol prior to instillation.</li>
		<li>Lubricate the catheter tip with normal saline. Put the index finger of the non-dominant hand on the abdomen and push it gently upwards. Start the catheterization of the urethra in a 90&#176; angle (vertically) and once resistance is encountered, tilt it horizontally before inserting it further (0.5 cm). 
		NOTE: Never forcefully push the catheter, as this will cause harm to the urethra. Gentle turning motions can be helpful in catheter insertion. On the other hand, a lack of resistance usually indicates erroneous insertion into the vagina.</li>
		<li>Perform a slow (5 &#181;L/s) instillation of 50 &#181;L of the bacterial inoculum (2 x 107 CFU). 
		NOTE: Higher volumes or faster instillation might cause reflux to the kidneys. During practice of the technique, blue ink can be used to evaluate reflux.</li>
		<li>After the instillation, keep the syringe and catheter in place for a few more seconds and then slowly retract to prevent leakage. Record any irregularities such as a high amount of leakage or a bloody meatus and exclude animals, if necessary.</li>
		<li>Position the animal in supine position at the nose cone of the imaging chamber and repeat the preceding steps 2.1.1-2.1.6 for all the remaining animals. Use one catheter per experimental group. Ensure anesthesia is continued and minimize the time between the first and the last animal.</li>
		<li>If necessary, administer antibiotics or experimental drugs, prior to or after the imaging (step 3). For example, to administer enrofloxacin, add 40 &#181;L of the enrofloxacin (100 mg/mL) solution to 3.96 mL of physiological saline to obtain a 1/100 dilution. Inject 100 &#181;L/10 g subcutaneously at 9 am and 5 pm to administer 10 mg/kg of enrofloxacin twice daily for 3 days.</li>
	</ol>
	</li>
</ol>
3. Bioluminescence imaging
<ol>
	<li>Preparation and selection of imaging parameters
	<ol>
		<li>Open the BLI acquisition software (see Table of Materials) and click on Initialize in the imaging device (see Table of Materials) to test the camera and stage controller system and to cool the CCD camera to -90 &#176;C. 
		&#8203;NOTE: During this process, the door is locked, and the progress of the initialization can be followed on the control panel. A green light indicates that the temperature of -90 &#176;C is reached. A warning will appear if imaging is attempted before completion of the initialization.</li>
		<li>Ensure data is saved automatically: Click on acquisition Auto-save to and select the correct folder.</li>
		<li>Select Luminescence and Photograph. Check the default luminescence settings: Set excitation filter to Block and emission filter to Open.</li>
		<li>Set the exposure time to Auto when taking the first image, especially when expecting a dim signal to ensure an adequate number of photon counts. For in vivo measurements and bright signals, set the Exposure Time to ~30 s. If the image is saturated, a warning will appear. If this happens, reduce the exposure time.</li>
		<li>Select the medium Binning, F/stop 1 and choose the correct field of view (FOV) (D for 5 animals).</li>
		<li>Set the subject height to 1 cm when imaging mice.</li>
		<li>Click on Add three times in the Acquisition Control Panel to obtain a sequence of three images, as technical replicates.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Place mice in the imaging chamber in the supine position and use the manifold nose cone to maintain anesthesia (isoflurane 1.5%) throughout the experiment. Image up to 5 mice simultaneously and separate the animals using the light baffle to prevent reflection.</li>
		<li>Close the door and click on Acquire to start the imaging sequence.</li>
		<li>Fill in detailed information about the experiment (wild type and knockout animals, treatments, day of imaging, etc.). The imaging settings, such as exposure time, are saved automatically.</li>
		<li>Remove mice from the imaging chamber and return them to their cage. Check for the full recovery after anesthesia. Within minutes, the animals should be fully awake and explorative. Do not provide analgesia as this might interfere with the UTI course18.</li>
		<li>Return the cages to the ventilated racks until the next imaging cycle. After completion of the experiment, euthanize the animals by CO2 asphyxiation or cervical dislocation. Do this before the recovery of isoflurane anesthesia to minimize distress.</li>
	</ol>
	</li>
	<li>Analysis of the images
	<ol>
		<li>Start the imaging software and load the experimental file by clicking on Browse.</li>
		<li>Use the Tool Palette to adjust the color scale of the image. Standardize the visual aspect of the results by using the same settings for all the images, i.e., use a logarithmic scale with a minimum of 104 to a maximum of 107 photons. These adjustments do not affect the raw data but only the graphical presentation.</li>
		<li>Use the ROI tools to draw a region of interest (ROI) on the image. Ensure the ROI is large enough to cover the complete area and use the same dimensions for all the images. Here, a square ROI of 3.5 cm x 4.5 cm was placed over the lower abdomen.</li>
		<li>Click on ROI measurement to quantify the light intensity (counts or total photon flux). Export these data and use the average of the technical replicates. 
		NOTE: Counts represent the number of photons detected by the camera and should only be used for the image quality control. To report data, use total photon flux. Total photon flux is more physiologically relevant as it represents the light emitted from the surface and it is a calibrated unit, which is corrected for the exposure time (per second).</li>
	</ol>
	</li>
</ol>

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The authors declare no conflicts of interest.

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...

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 sensitivity2,3,4. In recent years, there has been a growing interest in novel therapies for the treatment and prevention of recurrent UTI. Despite a better understanding of the innate and adaptive immunity of the lower urinary tract and of the bacterial virulence factors necessary for invasion and colonization, no radical changes in the treatment regime have been translated to the daily urological practice2. In order to study UTI pathogenesis and susceptibility in vivo, a reproducible and non-invasive assay is indispensable.
Multiple animal UTI models have been described ranging from nematodes to primates, but the murine model is predominantly used5,6. This model consists of transurethral catheterization of (female) mice and subsequent instillation of a bacterial suspension, most commonly uropathogenic Escherichia coli (UPEC), directly into the bladder lumen7. After inoculation, the bacterial load has traditionally been quantified by determining colony forming units (CFU). This technique requires sacrificing animals to obtain post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. Moreover, longitudinal follow-up of the bacterial load in individual animals is not possible using this technique.
In 1995, Contag et al. suggested the use of bioluminescent-tagged pathogens to monitor disease processes in living animals8,9. Since then, bioluminescence imaging (BLI) has been applied to numerous infection models such as meningitis, endocarditis, osteomyelitis, skin, and soft tissue infections, etc.10,11,12. In the murine UTI model, a UPEC strain with the complete lux operon (luxCDABE) from Photorhabdus luminescens can be used13. An enzymatic reaction is catalyzed by the bacterial luciferase which is dependent on the oxidation of long-chain aldehydes reacting with reduced flavin mononucleotide in the presence of oxygen, yielding the oxidized flavin, a long-chain fatty acid and light12. The lux operon encodes for the luciferase and other enzymes required for the synthesis of the substrates. Therefore, all metabolically active bacteria will continuously emit blue green (490 nm) light without the need for the injection of an exogenous substrate12. Photons emitted by lux-tagged bacteria can be captured using highly sensitive, cooled charge-coupled device (CCD) cameras.
The use of bioluminescent bacteria in a model for UTI allows for the longitudinal, non-invasive quantification of the bacterial load, omitting the need for sacrificing animals at fixed time points during the follow-up for CFU determination. Despite the wide range of possibilities, accumulating evidence for the robustness of this BLI technique in other fields and its advantages over classic models of UTI, it has not been widely implemented in the UTI research. The protocol presented here provides a detailed step-by-step guide and highlights the advantages of BLI for all future UTI research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well Black Flat Bottom Polystyrene Plate</td>
			<td>Corning</td>
			<td>3925</td>
			<td>for in vitro imaging</td>
		</tr>
		<tr>
			<td>Aesculap ISIS</td>
			<td>Aesculap</td>
			<td>GT421</td>
			<td>hair trimmer, with GT608 cap</td>
		</tr>
		<tr>
			<td>Anesthesia vaporizer</td>
			<td>Harvard apparatus limited</td>
			<td>N/A</td>
			<td>https://www.harvardapparatus.com/harvard-apparatus-anesthetic-vaporizers.html</td>
		</tr>
		<tr>
			<td>Baytril 100 mg/mL</td>
			<td>Bayer</td>
			<td>N/A</td>
			<td>Enrofloxacin</td>
		</tr>
		<tr>
			<td>BD Insyte Autoguard 24 GA</td>
			<td>BD</td>
			<td>382912</td>
			<td>Yellow angiocatheter, use sterile plastic tip for instillation</td>
		</tr>
		<tr>
			<td>C57Bl/6J mice</td>
			<td>Janvier</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5804R</td>
			<td>Eppendorf</td>
			<td>EP022628146</td>
			<td></td>
		</tr>
		<tr>
			<td>Dropsense 16</td>
			<td>Unchained Labs</td>
			<td>Trinean</td>
			<td>to measure OD 600nm</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline, Gibco</td>
			<td>ThermoFisher Scientific</td>
			<td>REF 14040-083</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70% denaturated 5L</td>
			<td>VWR international</td>
			<td>85825360</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 14ml Round Bottom Polystyrene Tube, Snap-Cap</td>
			<td>Corning</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50ml cellstart</td>
			<td>Greiner</td>
			<td>227285</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton GASTIGHT syringe, PTFE luer lock, 100 &#181;L</td>
			<td>Sigma-Aldrich</td>
			<td>26203</td>
			<td>to ensure slow bacterial instillation of 50 &#181;L</td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Roth</td>
			<td>6174.1</td>
			<td>holder: Art. No. 6189.1</td>
		</tr>
		<tr>
			<td>Iso-Vet 1000mg/g</td>
			<td>Dechra Veterinary products</td>
			<td>N/A</td>
			<td>Isoflurane</td>
		</tr>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>REF 124262</td>
			<td>imaging device</td>
		</tr>
		<tr>
			<td>Kanamycine solution 50 mg/mL</td>
			<td>Sigma-Aldrich</td>
			<td>CAS 25389-94-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Imaging Software</td>
			<td>PerkinElmer</td>
			<td>N/A</td>
			<td>BLI acquisition software, version 4.7.3</td>
		</tr>
		<tr>
			<td>Luria Bertani Broth</td>
			<td>Sigma-Aldrich</td>
			<td>REF L3022</td>
			<td>alternatively can be made</td>
		</tr>
		<tr>
			<td>Luria Bertani Broth with agar</td>
			<td>Sigma-Aldrich</td>
			<td>REF L2897</td>
			<td>alternatively can be made</td>
		</tr>
		<tr>
			<td>Petri dish Sterilin 90mm</td>
			<td>ThermoFisher Scientific</td>
			<td>101VR20</td>
			<td>to fill with LB agar supplemented with Km</td>
		</tr>
		<tr>
			<td>Pyrex Culture flask 250 mL</td>
			<td>Sigma-Aldrich</td>
			<td>SLW1141/08-20EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide 200 Trinean</td>
			<td>Unchained Labs</td>
			<td>701-2007</td>
			<td>to measure OD 600nm</td>
		</tr>
		<tr>
			<td>UTI89-lux</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Generous gift from Prof. Seed</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR international</td>
			<td>444-1372</td>
			<td></td>
		</tr>
	</tbody>
</table>

longitudinal follow-up of urinary tract infections and their treatment in mice 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.

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). ...

Watch this Scientific Journal Video about Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging at JoVE.com

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

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, ...

longitudinal-follow-up-urinary-tract-infections-their-treatment-mice

Research

JoVE Journal

Immunology and Infection

2.8K Views.  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.

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

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...