Transurethral Catheterization and Bacterial Inoculation to Generate a Urinary Tract Infection Model in Male Mice

Abstract

Source: Zychlinsky Scharff, A., et al. Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice. J. Vis. Exp. (2017).

This video demonstrates the establishment of uropathogenic Escherichia coli infection in a female mouse bladder through transurethral catheter instillation. The injected bacteria successfully establish a urinary tract infection within the bladder, providing a valuable platform for studying healthcare-associated diseases related to the urinary tract.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Catheters

1. Prepare one pediatric intravenous-access cannula for each group of mice to be infected. Using the inbuilt spring mechanism, divest each cannula of its needle, as instructed by the manufacturer. Discard the needles, preserving only the plastic intravenous cannula.
2. Sterilize catheters in a laminar flow hood for one ultraviolet (U.V.) cycle, typically 25 – 30 min.
   Note: Catheters can be used for more than one mouse, however, a new catheter should be used between experimental groups, sexes, or bacterial strains.

2. Preparation of Uropathogenic E. coli for Infection

1. At least two days prior to infection, using a sterile inoculation loop, streak UPEC from a frozen bacterial stock onto a Luria Broth (LB) agar plate, containing antibiotics if appropriate. Incubate at 37 °C overnight. Plates can be stored at 4 °C for up to one week.
2. In a 100 ml sterile Erlenmeyer flask, inoculate 10 ml LB, containing antibiotics if appropriate, with a single colony from the LB plate and incubate standing at 37 °C overnight (approximately 16 – 18 hr).
   Note: Standing cultures permit the expression of type 1 pili, which are necessary for infection. Cultures grown shaking will have less efficient pili expression, and therefore, inconsistent rates of infection.
3. Measure the optical density (OD)₆₀₀ of the overnight culture and calculate the number of bacteria per ml using a pre-determined growth curve. As an example, for UPEC strain UTI89, OD₆₀₀ = 0.35 is equivalent to 2 x 10⁸ CFU per ml, thus, an overnight culture with an OD₆₀₀ = 2.4 would be equivalent to 13.7 x 10⁸ CFU per ml. Empirically determine the relationship between the OD₆₀₀ and viable bacteria with every new strain employed for infection.
4. Determine the amount of bacteria needed by considering the number of animals to be infected and that each mouse should receive approximately 10⁷ colony-forming units (CFU) in 50 μl PBS. Include in the calculation the ~130 µl dead volume of the catheter and syringe nub and 100 µl needed to determine the inoculum.
   1. Spin the bacterial suspension in a tabletop microcentrifuge at 17,000 x g for 1 min and resuspend the resulting bacterial pellet at 2 x 10⁸ CFU per ml in PBS. Serially dilute an aliquot of this suspension and plate it on LB agar, with antibiotics if appropriate, to determine the exact inoculum for each infection.
5. Draw the bacterial inoculum into a 1 ml syringe and attach the catheter to the end of the syringe. Tap the syringe to remove any air and depress the plunger to fill the dead air space in the catheter before beginning the instillation.

3. Preparation of Mice

1. Anesthetize mice via intraperitoneal injection of 100 mg/kg ketamine and 5 mg/kg xylazine. Supplemental heat can be provided.
2. Ensure that each mouse is fully sedated by squeezing the footpad with medium pressure. Mice are fully sedated when they do not react and require 3 – 5 min to reach this state.
3. Place mice supine and apply medium pressure to the lower abdomen to empty the bladder of urine. Full bladders feel like a pea under the skin between the iliac crests.
   Note: Inhaled isoflurane has been used to catheterize female mice, however, it has not been tested whether male mice are sufficiently anesthetized by inhaled isoflurane for this procedure.

4. Transurethral Instillation of Male Mice

1. Place two thumb forceps cranially and caudally to the mouse's external genitalia. Retract the prepuce to fully expose the glans penis. Once the penis is externally positioned, release the thumb forceps.
2. Reposition the forceps to stabilize the protruding penis perpendicular to the animal, holding the organ gently but tautly. Visualize the urethral meatus and carefully introduce the catheter into the small opening at the tip of the organ. Gently guide the catheter into the penis, toward the body of the mouse, maintaining gentle tension with the forceps. The catheter requires no lubrication. Do not force the catheter into the penis; the catheter should slide smoothly into the urethra, indicating correct placement.
3. Once the hub of the catheter meets the tip of the penis, very slowly dispense 50 µl of the inoculum, while maintaining the position of the penis. The quality of the instillation can be noted at this time according to a predetermined instillation quality score, such as that shown in Figure 1.
4. Retract the catheter slowly, over a count of 5, to prevent leakage of the inoculum. Place mice in their cages in a supine position. Animals should begin to recover 30 – 45 min following administration of the anesthetic.

Representative Results

Figure 1: The Quality of Instillation does not Correlate with Bacterial Burden at 24 hr Post-infection. Six to 8 week-old female and male C57Bl/6 mice were instilled with 10⁷ CFU of UPEC. Immediately after each instillation, a single researcher assigned a quality score to the instillation, as defined by specific criteria (boxed text). 24 hr post-infection, bladders were aseptically removed to enumerate bacterial burden. The plot depicts the assigned quality score versus CFU/bladder. Each dot is one mouse, and 5 pooled experiments are shown. p = 0.17, Kruskal-Wallis test comparing the mean rank of each column with the mean rank of every other column with post hoc Dunn's test for multiple comparisons.

Materials

BD Insyte Autoguard Shielded IV Catheters 24 G, 0.7 mm external diameter, 14 mM long	BD Medical	381811 or 381411*	* catalog number is country dependent
Inoculating loop	Greiner Bio-One	731171
syringes	B Braun	9166017V
Thumb (Adson) forceps	Fine Science Tools	11006-12
insulin syringe	Terumo	BS05M2913
5 ml tubes, polypropylene	Falcon	352063
LB Miller broth	Difco	244620
LB Miller agar	Difco	244520
Cuvettes	Bio-rad	223-9955