Establishment and Characterization of UTI and CAUTI in a Mouse Model

The overall goal of this procedure is to display a reliable method for the establishment of urinary tract infections or UTIs and catheter associated UTIs, or ca a UTI I in a mouse model, and to assess the infections. This is accomplished by first properly preparing the bacterial inoculums, the catheter and needle. Next, the mouse urinary tract is intravesical, inoculated, and catheterized.

Then the urinary tract tissues and catheters are harvested to evaluate the infection. Finally, the infection is quantified by determining bacterial tissue, urine, and catheter load and or intracellular bacterial community or IBC formation. Ultimately, CFU enumeration and laxy staining are used to show the establishment persistence and severity of urinary tract colonization within a mouse model.

The main advantage of this technique is to develop a reliable model for mimicking human UTIs in order to uncover critical steps in TI pathogenesis. This model has aided in the development of novel treatments and therapeutics for UTIs. Visual demonstration of this method is critical as the inoculation, ization and assessment of intracellular bacterial communities are difficult to learn because the manipulation of the urethra and the bladder tissue are not intuitive.

To begin thread approximately one inch of PE 10 tubing onto the shaft of a 30 gauge needle. UV sterilize overnight and prepare bacterial inoculum. According to the text protocol on a sterilized workstation, draw up to 0.9 milliliters of the prepared bacterial inoculum into a one milliliter syringe.

Attach the prepared sterile inoculum needle with PE tubing onto the syringe, and with sterile scissors, trim the tubing to one millimeter in length. After anesthetizing a female mouse according to the text protocol, place it on its back on a paper towel and spread the legs. Attach a nose cone to the mouse and provide a controlled supply of iso fluorine.

Gently palpate the bladder to induce urination and ensure avoided bladder and use a 100%ethanol wipe to clean the periurethral area. Dab the inoculation needle syringe point first into the surgical lubricant. Then insert the inoculation needle into the urethra approximately 12 millimeters and gently depress the syringe plunger to dispense 50 microliters of the bacterial solution into the platter.

To collect urine, pre or post infection, gently restrain the mouse by holding the tail and placing it on a flat elevated surface such as the top of a mouse cage. Hold a sterile 1.5 milliliter tube under the mouse urethra and gently press down on the back of the mouse near the tail to apply pressure to the bladder. Catch the urine in the tube.

After preparing serial dilution of the urine and culturing overnight, count the colonies to carry out the C-A-U-T-I protocol. Begin by cutting a seven millimeter piece of PE 10 tubing and a five millimeter piece of silicone tubing such as reil. Remove the cap of a 30 gauge needle and use the PE 10 tubing to thread the needle up to the base.

Then feed the silicone tubing onto the PE 10. To implant the catheter, attach the catheter needle to an empty one milliliter syringe. Cut a one inch square piece of paraform and put a dab of surgical lubricant on top.

After anesthetizing the mouse, place the mouse on its back on a paper towel and attach a nose cone to deliver isof fluorine. Gently palpate the bladder to induce urination and ensure avoided bladder. Then use a 100%ethanol wipe to clean the periurethral area.

Next, use 10%povidone iodine solution and a cotton tipped applicator to disinfect the periurethral area, dab the tip of the inoculation needle into the surgical lubricant. Then insert the catheter into the urethral opening. Once in use tweezers to push the PE tubing toward the bladder, which will deposit the smaller silicone catheter into the bladder.

Then immediately remove the needle with the seven millimeter tubing still attached. After preparing EFAs bacterial inoculum, use it to inoculate the bladder and return the animal to its cage before determining the bacterial burden as per the following demonstration. Once the animal has been euthanized, place it on its back and use 70%ethanol to spray the abdomen, dissect the mouse, harvesting the bladder, and then kidneys in that order, and place the organs into separate five milliliter tubes containing one XPBS.

If the mouse is catheterized, remove the catheter from the bladder and place it in a separate tube. After homogenizing the organs preparing serial dilution and culturing overnight on LB aerates, count the colonies for IBC enumeration. After harvesting the bladder, place it in a silicone coated well of a six well plate with PBS and use scissors to cut it in half.

Using small metal pins placed along the outermost edges, gently splay the bladder so that the lumen is facing upward and the maximum amount of urothelium is exposed. After performing XI stain as outlined in the text protocol, observe IBCs, which appear as blue puncta under a dissecting microscope. The data shown here represent acute stage urinary tract colonization by the prototypical up e cystitis isolate UTI 89.

In this figure, CFU occurring four weeks after inoculation of C3 HHEN mice with two times 10 to the seven CFUs of U UTI I 89 are shown Under these experimental conditions, 20 to 50%of infected mice develop chronic cystitis while the remaining 50 to 80%of mice resolve the infection IBC quantification is an informative measure of bacterial burden during acute cystitis and high levels of IBCs correlate with the risk of developing chronic cystitis. As seen here using LAE becyase staining IBCs appear as blue purple puncti on the urothelium that can be counted in the C3 HHEN background. An average of about 50 IBCs can be seen per bladder.

The data shown here are from a bladder implant mouse model of CA A UTI and represent 24 hour infections with epha list strain OG one rf. When one times 10 to the seven CFUs of OG one RF are intravesical inoculated into an unplanted bladder, the infection begins to clear by 24 HPI and as resolved by 3D PI.Conversely, OG one RF inoculated into an implanted bladder results in colonization of both the catheter and the bladder with tenfold higher colonization levels at 24 HPI than in unplanted bladders. Following this procedure, other techniques such as confocal and electron microscopy can be used to further evaluate IBC formation and bacterial localization.

After its development, this technique fade a wave of many researchers in the UTI field to explore bacterial virulence factors and host responses in mice that recapitulates the human pathogenesis.