Transurethral Induction of Mouse Urinary Tract Infection

Summary

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

Abstract

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°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.

Protocol

I. Preparation of Urethral Catheters

1. For each mouse bladder and kidney to be sampled, place 5-6 stainless steel beads (bladder, 1.6 mm size or 0.9-2.0 mm blend) or zirconium oxide beads (kidney, 0.5 mm size) in one cryovial tube, respectively. Autoclave the bead-containing tubes.
2. After the tubes cool to ambient temperature, add 200 microliters of sterile PBS to each stainless steel bead-containing tube and 400 microliters of sterile PBS to each zirconium oxide bead-containing tube.
3. We prepare urethral catheters as described by Hung and Hultgren¹³. Autoclave a clean pair of flat-head forceps and fine scissors (iris or sim....

Discussion

Induction of mouse urinary tract infection by transurethral inoculation of bacteria is a long-established but technically demanding procedure¹¹. Hung and Hultgren recently published an excellent review of transurethral techniques of mouse urinary tract infection¹³. In this paper, we attempt to further illustrate the finer points of this experimental approach.

Novices may practice their technique by performing a midline laparotomy to expose the bladder, injecting a dilu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cryovial tubes, 2.0 ml polypropylene (no caps)	E&K Scientific	640201
Caps for cryovial tubes, with ’O’ ring	E&K Scientific	440011-N
Bullet Blender	Next Advance	BBX24
Zirconium Oxide Beads (0.5mm)	Next Advance	ZROB05
Stainless Steel Beads (1.6mm)	Next Advance	SSB16
Stainless Steel Bead Blend (0.9-2.0mm)	Next Advance	SSB14B
CBA/J Mice, 8-12 week old female	Jackson Laboratory	000656
Phosphate Buffered Saline (NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO4 1.4mM)	EMD Millipore	EM-SX0420-13 (NaCl) EM-PX1405-1 (KCl) EM1.06585.0 (Na2HPO4) EM-5108-1 (KH2PO4)
LB agar
LB broth	EMD Millipore	1.10285.0500
Isoflurane (Aerrane)	Baxter Internationl Inc.	NDC 10019-773-60
Inoculating loops	Fisher Scientific	22-363-601
Eosin-methylene blue (EMB) agar powder	Himedia Laboratories	MM022-500G
MacConkey agar powder	Difco Laboratories	212123
LB agar powder	Difco Laboratories	244520
Intramedic non-radiopaque polyethylene tubing, PE10 [inner dimension 0.28 mm (0.011 inch); outer dimension 0.61 mm (0.024 inch)]	BD Biosciences	427401
30 gauge needles, 1/2 inch	BD Biosciences	305106
1 ml tuberculin Slip-Tip syringe	BD Biosciences	309602
3 ml Luer-Lock syringe	BD Biosciences	309585