Catheter-Associated Urinary Tract Infection in a Mouse Model

Abstract

Source: Conover, M. S., et al., J. Establishment and Characterization of UTI and CAUTI in a Mouse Model. J. Vis. Exp. (2015)

This video demonstrates the procedure for assembling and implanting a transurethral catheter in a female mouse, followed by bacterial inoculation to establish catheter-associated urinary tract infection and investigate biofilm-mediated host-pathogen interactions.

Protocol

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

1. CAUTI (Catheter-associated urinary tract infection) Model Protocol, Catheter Needle Preparation for CAUTI Model

1. Remove the cap of the 30 G needle. Cut a 7 mm piece of PE10 (polyethylene 10) tubing and 5 mm piece of silicone tubing such as RenaSIL. Thread the needle with PE10 tubing until the tubing touches the base of the needle.
2. Then feed the 5 mm piece onto the PE10-containing needle. UV (ultraviolet) sterilize the needle assemblies overnight.
   NOTE: When loading the silicone tubing, leave approximately 1 mm of the tubing extending past the needle tip.

2. E. faecalis OG1RF Bacterial Inoculum Preparation

1. Prepare OG1RF inoculum starting 48 hr prior to the inoculation day. Streak OG1RF onto a BHI (brain heart infusion) plate from a frozen stock.
2. Pick a colony and inoculate it into 10 ml of BHI and grow it statically for 18 hr at 37 °C. Centrifuge the culture and wash the pellet (3 times) in 1 ml volumes of sterile 1x PBS.
3. Resuspend the bacterial pellet in 1x PBS (phosphate buffered saline). Measure bacterial optical density and adjust the concentration to the desired inoculum size. The standard concentration of the inoculum used is 10⁷; however, this may vary due to the design of the study

3. Catheter Implantation

1. Clean the workstation with 70% ethanol and cover the area with an absorbent cover (or use a sterile flow hood).
2. Attach catheter-needle to an empty 1 ml (TB or tuberculin) syringe. Cut a 1 inch square piece of parafilm and put a dab of surgical lubricant (approximately the size of a dime) on top.
   NOTE: Two different needles are used for deposition of the catheter and for the inoculum to minimize accidental inoculation due to the mechanical manipulation necessary for catheter implantation. This also allows for the convenience of preparing fewer inoculation needles.
3. Anesthetize C57BL/6 mice by putting them in a 32-ounce glass jar containing a tea-infuser ball with cotton balls soaked in 3 ml of isoflurane or a vaporizer chamber (following the manufacturer's protocol) until unconscious but still breathing normally (1 breath/sec).
4. Then put the mouse on its back on a paper towel and spread its legs. Cover the nose of the mouse with a nose cone or a 50 ml conical tube with cotton balls containing a small amount (approximately 1-2 ml) of isoflurane.
   CAUTION: Isoflurane is an inhalation anesthetic. Use in a well-ventilated area and minimize inhalation by the researcher.
5. Gently palpitate the bladder to induce urination and ensure a voided bladder. Wipe the periurethral area with a 100% ethanol wipe.
6. Disinfect the periurethral area with 10% povidone-iodine solution using a cotton-tip applicator. Dab the inoculation needle/syringe, point first, into the surgical lubricant.
   NOTE: Povidone-iodine is used for catheter implantation, which is a stronger aseptic solution than alcohol. Catheter implantation requires more mechanical manipulation to insert the catheter and thus has a greater potential for contamination.
7. Insert the catheter into the urethral opening. Once in, use tweezers to push the (7 mm PE10) long piece toward the bladder, consequently pushing the (5 mm silicone) small catheter and depositing it into the bladder. Remove the needle, with the 7 mm tubing still attached, immediately.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material for catheter and needle preparation:
30 G needles	BD Precision Glide	305106	30 G x ½ (0.3 mm x 13 mm)
PE10 polyethylene tubing	BD	427400	Inside diameter -0.011 in (0.28 mm); outside diameter – 0.024 in (0.61 mm)
RenaSIL 025 platinum cured silicon tubing	Braintree Scientific, Inc	SIL 025	inside diameter-0.012 x outside diameter 0.025, 25 ft coil
Material for infections:
Isoflourane – Isothesia	Butler Schein	29405	250 ml
Clear Glass Straight-Sided Jar	Kimble Chase	5413289V 21
Stainless Steel Tea Infuser	Schefs-Amazon		Premium Loose Leaf Tea Infuser By Schefs - Stainless Steel - Large Capacity -
Non-sterile cotton balls	Fisherbrand	22-456-880
50 ml Falcon tubes	VWR	89039-660
Isotec 3 -vaporizer	Ohmeda	1224478
Ear punch	Fisher Scientific	13-812-201	(when necessary)
Betadine solution	Betadine solution		10% Povidie-iodine topical solution
Q-tips	Fisher Scientific	22-037-924	6 inches
Diapers for bench	Fisherbrand	14206 63	Absorbent Underpads (20”X36”mats)
Surgical lubricant	Surgilube	0281-0205-36
Dissecting scissor	Fine Science tools, INC	14084-08
Micro-Adson Forceps	Fine Science tools, INC	11018-12
1 ml syringe	BD	309659	Tuberculin slip tip
Parafilm	Bemis	PM996	4 inches x 125 FT
Eppendorf rack	Fisherbrand	05-541-1
Eppendorf tubes	MIDSCI	AVX-T-17-C
Harvesting catheters, bladders and kidneys:
Homogenizer	PRO Scientific INC	Bio-Gen Pro 200
5 ml polypropylene round-bottom tube	BD	352063	for organ homogenization
Paper towel	Georgia-Pacific
Ethanol	Pharmco-AAPER	11100020S	200 proof
Costar™ Clear Polystyrene 96-Well Plates	Corning	3788
1x Phosphate sodium saline	Sigma-Aldrich	P3813
BRANSONIC Ultrasonic cleaner 1210	Branson Ultrasonics Corporation	1210