The enumeration of colony forming units was limiting the reproducibility of research in the UTI field. Bioluminescence imaging will advance in vivo UTI research on bladder physiology, UTI pathogenesis and susceptibility. Bioluminescence imaging enables longitudinal follow up with real-time insight into the evolution of the infection.
Moreover, it drastically reduces the number of animals needed. Bioluminescence imaging facilitates research on recurrent or chronic infections, which are frequent in humans. Additionally, researchers can identify ascending infections or dissemination to the blood using bioluminescence imaging.
Obtain single colonies by streaking out the glycerol stock of bacteria with an inoculation loop on LB plates supplemented with kanamycin sulfate, and culturing overnight at 37 degrees Celsius. Fill a sterile 14 milliliter polystyrene round bottom tube, with dual-position snap caps with five milliliters of LB broth supplemented with kanamycin sulfate. Pick a single bacterial colony with an inoculation loop, and add this to the LB broth.
Vortex for 10 seconds to ensure proper mixing. Culture statically with the snap cap in the open position at 37 degrees Celsius overnight. After the incubation, vortex the tube for 10 seconds to ensure proper homogenization of the bacterial culture.
Make a subculture in the Erlenmeyer by adding 25 microliters of the bacterial suspension to 25 milliliters of fresh LB medium without antibiotics. Close the Erlenmeyer and culture statically overnight. On the day of installation, pour the culture from the Erlennmeyer into a 50 milliliter culture tube, and centrifuge.
Decant the supernatant and resuspend the bacterial pellet in 10 milliliters of sterile PBS. Vortex the tube again for 10 seconds. Choose the experimental concentration of the inoculum and determine the corresponding OD at 600 nanometers using the standard curve.
Addd one milliliter of the resuspended bacterial culture into nine milliliters of sterile PBS and adjust until the desired OD 600 nanometer is reached. Mount a sterile 24-gauge angiocatheter tip on a 100-microliter syringe. Fill the syringe with the prepared bacterial solution.
Place one animal on a working surface in the supine position and maintain a stable isoflurane anesthesia using a nose cone during the installation. Apply the eye ointment. Expel the residual urine by applying gentle compression and making circular movements on the suprapubic region, then clean the lower abdomen with 70%ethanol.
Lubricate the catheter tip with normal saline. Put the index finger on the non-dominant hand on the abdomen and push it gently upwards. Start the catheterization of the urethra vertically in a 90-degree angle.
Once resistance is encountered, tilt it horizontally before inserting it further. Perform a slow installation of 50 microliters of the bacterial inoculum. After the installation, keep the syringe and catheter in place for a few more seconds and then slowly retract to prevent leakage.
Use one catheter per experimental group. Position the animal in a supine position in the nose cone to prepare it for imaging, and prepare it for imaging using one catheter per experimental group. If necessary, administer antibiotics or experimental drugs, as mentioned in the text manuscript.
Open the BLI acquisition software and click on Initialize in the imaging device to test the camera and stage controller system and to cool the charge-coupled device camera to minus 90 degrees Celsius. Click on Acquisition, AutoSave To.Select Luminescence and Photograph. Check the default luminescent settings by setting the excitation filter to Block and emission filter to Open.
Set the exposure time to auto when taking the first image. For in vivo measurements and bright signals, set the exposure time to around 30 seconds. Reduce the exposure time if a warning appears due to a saturated image.
Select the medium binning, F/stop one, and choose the correct FOV. Set the subject height to one centimeter when imaging mice. Image up to five miles simultaneously and separate the animals using the light baffle to prevent reflection.
Close the door and click on Acquire to start the imaging sequence, then fill in detailed information about the experiment. Remove mice from the imaging chamber and return them to their cage. Check for full recovery after anesthesia, then return the cages to the ventilated racks until the next imaging cycle.
Start the imaging software and load the experimental file by clicking on Browse. Use the tool palette to adjust the color scale of the image. Use the ROI tools to draw a region of interest on the image, ensuring that it is large enough to cover the complete area and using the same dimensions for all the images.
Then click on ROI measurement to quantify the light intensity. Subsequent images of mice obtained immediately post-installation showed that was robustly detectable above 20, 000 colony forming units, and a linear correlation between the colony forming units of the inoculum and the bioluminescence in vivo was established. The natural evolution of a urinary tract infection was studied in a control group that did not receive any treatment.
The effect of antibiotic treatment on the infection kinetics was visualized in detail in a group of antibiotic-treated animals. An immediate decrease in bacterial load, as measured by total photon flux, was seen after the first dose of Enrofloxacin. None of the treated animals had a subsequent rise in bacterial load.
The overall bacterial load of each animal was calculated using the area under the curve of the log transformed total photon flux, showing a significant difference between animals treated with Enrofloxacin and untreated animals over the time course of 10 days. These results provide proof of concept that BLI can be used to evaluate differences in UTI infection kinetics. Bioluminescence imaging is a noninvasive technique that can be combined with other methods such as regular collection of urinary samples for bacterial or biochemical analysis or for the experiments.
Here we have demonstrated that bioluminescence imaging is a powerful tool to evaluate novel therapeutic strategies on the disease course of urinary tract infections.
