The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work’s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.
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Method Article
Establishment and Characterization of UTI and CAUTI in a Mouse Model
In This Article
Erratum Notice
Important: There has been an erratum issued for this article. View Erratum Notice
Summary
Abstract
Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.
Introduction
Urinary tract infections (UTIs) are one of the most common bacterial infections and can be divided into two categories based on the mechanism of acquisition, community and nosocomial acquired UTI. Community-acquired UTIs often occur in otherwise healthy women and studies have shown that approximately 50% of women will have at least one UTI in their lifetime 1. Additionally, recurrence is a major problem. A woman who has an initial acute infection has a 25-40% chance of having a second infection within six months despite appropriate antibiotic treatment and many women continue to have frequent recurrences 2. The bacteria that cause these infection....
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Protocol
Ethics statement: The Washington University Animal Studies Committee approved all mouse infections and procedures as part of protocol number 20150226, which expires 12/10/2018. Overall care of the animals was consistent with The Guide for the Care and Use of Laboratory Animals from the National Research Council and the USDA Animal Care Resource Guide. Euthanasia procedures are consistent with the “AVMA guidelines for the Euthanasia of Animals 2013 edition.”
1. UPEC UTI Protocol, Inoculation Needle Preparation (Figure S1)
- Remove the cap of the 30 G needle. Thread approximately 1 inch of PE10 tubing onto the shaft of t....
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Results
The intravesical models of uncomplicated and catheter associated UTI provide flexible platforms for elucidating the molecular mechanisms of bacterial pathogenesis, the impact of these diseases on host tissue, and the development and testing of novel approaches to manage these common and costly infections. Depending on the mouse strain and pathogen, intravesical inoculation can be used to study host-pathogen interactions to elucidate factors necessary for initiating or modulating acute (Figure 1 and
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Discussion
Uncomplicated community acquired UTI is a common and costly infection accounting for several million primary care visits every year 46. In addition, CAUTIs are a common healthcare acquired infection that has become extremely costly to healthcare providers as the Centers of Medicare and Medicaid Services no longer reimburses providers for the added cost of treatment resulting from hospital acquired CAUTI 45. The mouse models of UTI, both uncomplicated cystitis and CAUTI, described in these protocols .......
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Disclosures
The authors declare that they have no competing financial interests.
Acknowledgements
Funding for this work was provided by ORWH SCOR P50 DK064540, RO1 DK 051406, RO1 AI 108749-01, F32 DK 101171, and F32 DK 104516-01.
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Materials
| 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 | |
| IBC materials: | |||
| 6-well tissue culture test plate | Techno Plastic Products | 92006 | |
| Pins | Fine Science Tools | 26002-20 | |
| Sylgard 184 | Dow Corning | 3097358-1004 | Silicone Elastomer Kit |
| X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactoside) | Invitrogen | 15520-034 | Ultrapure |
| N, N-Dimethylformamide | Sigma Aldrich | D4551 | |
| MgCl2 (Magnesium chloride) | Sigma Aldrich | M8266 | |
| Sodium deoxycholate | Sigma Aldrich | D6750 | |
| Nonidet-P40 | Roche | 11754599001 | Octylphenolpoly(ethyleneglycolether)n |
| Potassium hexacyanoferrate(II) trihydrate (K-ferrOcyanide) | Sigma Aldrich | P3289 | |
| Potassium hexacyanoferrate(III) (K-ferrIcyanide) | Sigma Aldrich | 60299 |
References
- Foxman, B. Epidemiology of urinary tract infections: incidence, morbidity, and economic costs. Dis Mon. 49, 53-70 (2003).
- Foxman, B., et al. Risk factors for second urinary tract infection among college women. American journal of ....
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Reprints and Permissions
Erratum
Formal Correction: Erratum: Establishment and Characterization of UTI and CAUTI in a Mouse Model
Posted by JoVE Editors on 8/18/2017. Citeable Link.
An erratum was issued for: Establishment and Characterization of UTI and CAUTI in a Mouse Model. The Protocol section has been updated.
The ethics statement has been updated from:
Ethics statement: The Washington University Animal Studies Committee approved all mouse infections and procedures as part of protocol number 20120216, which was approved 01/11/2013 and expires 01/11/2016. Overall care of the animals was consistent with The guide for the Care and Use of Laboratory Animals from the National Research Council and the USDA Animal Care Resource Guide. Euthanasia procedures are consistent with the “AVMA guidelines for the Euthanasia of Animals 2013 edition.”
to:
Ethics statement: The Washington University Animal Studies Committee approved all mouse infections and procedures as part of protocol number 20150226, which expires 12/10/2018. Overall care of the animals was consistent with The Guide for the Care and Use of Laboratory Animals from the National Research Council and the USDA Animal Care Resource Guide. Euthanasia procedures are consistent with the “AVMA guidelines for the Euthanasia of Animals 2013 edition.”
Step 3 has been updated from:
3. Bacterial Inoculation
- Clean the workstation with 70% ethanol and cover area with absorbent paper (or use sterile flow hood).
- Draw up to 0.9 ml of the prepared bacterial inoculum into a 1 ml (TB) syringe (remove air bubbles). Attach a prepared sterile inoculation needle with PE10 tubing, onto the syringe containing the inoculum, then sterilely trim the polyethylene tubing.
NOTE: Leave 1 mm of tubing above the tip of the needle to avoid puncturing the bladder. - Cut a 1 inch square piece of parafilm and put a dab of surgical lubricant (approximately the size of a dime) on top.
- Anesthetize female C3H/HeN mice by putting them in a 32 ounce glass jar containing a tea-infuser ball with cotton balls soaked with 3 ml of isoflurane or vaporizer chamber (following manufactures protocol) until unconscious but still breathing normally (1 breath/sec).
NOTE: Some IACUC committees do not approve the use of a jar or vaporizer. Please follow the indication of the IACUC committee of your institution.
NOTE: If glass jar with tea-ball and/or nose cone with isoflurane-soakd cotton balls are used, the animal must be carefully observed to avoid anesthetic overdose and death. The advantage of using a vaporizer and anesthesia chamber is that it provides controlled isoflurane administration that avoids accidental overdoses. CAUTION: Isoflurane is an inhalation anesthetic. Use in a well-ventilated area and minimize inhalation. - Remove the mouse from the jar/vaporizer and place it on its back on a paper towel and spread the legs.
- Cover the nose of the mouse with a nose cone (a tube connected to the vaporizer that provides a controlled isoflurance dose that comes equipped on some vaporizer units) or 50 ml conical tube with a cotton ball containing a small amount (approximately 1-2 ml) of isoflurane.
- Gently palpitate the bladder to induce urination and ensure a voided bladder. Wipe the periurethral area with 100% ethanol wipe. Dab the inoculation needle/syringe, point first, into the surgical lubricant.
- Inoculate each mouse with 50 µl of the bacteria solution by inserting the inoculation needle transurethrally, approximately 12 mm, and pressing down on the syringe plunger gently to dispense the inoculum into the bladder gently (10 µl/sec). Remove the inoculation needle from the mouse.
NOTE: Immediate return of inoculum at the urethral opening when beginning to inoculate indicates improper or incomplete insertion of the needle. - Remove the mouse from nose cone and return it to its cage. Repeat steps 3.3-3.8 for each mouse. When/if switching inoculum conditions/strains, dispose of the syringe and inoculation needle in an approved sharps container and start again step 3.2.
NOTE: Inoculation with uropathogenic organisms generally does not cause severe pain symptoms. However, in rare instances, administering large doses of pathogens may cause fever, reduction in food and water intake and abnormal behavior. Animal health should be monitored throughout the experiment. If overt pain symptoms are notice, an analgesic, such as Buprenorphine (0.05−0.1 mg/kg given subcutaneously), can be applied. Procedures should be in accordance with each institution’s IACUC.
to:
3. Bacterial Inoculation
- Clean the workstation with 70% ethanol and cover area with absorbent paper (or use sterile flow hood).
- Draw up to 0.9 ml of the prepared bacterial inoculum into a 1 ml (TB) syringe (remove air bubbles). Attach a prepared sterile inoculation needle with PE10 tubing, onto the syringe containing the inoculum, then sterilely trim the polyethylene tubing.
NOTE: Leave 1 mm of tubing above the tip of the needle to avoid puncturing the bladder. - Cut a 1 inch square piece of parafilm and put a dab of surgical lubricant (approximately the size of a dime) on top.
- Anesthetize female C3H/HeN mice by putting them in a vaporizer chamber (following manufactures protocol) until unconscious but still breathing normally (1 breath/sec).
NOTE: Some IACUC committees do not approve the use of a vaporizer. Please follow the indication of the IACUC committee of your institution.
CAUTION: Isoflurane is an inhalation anesthetic. Use in a well-ventilated area and minimize inhalation. - Remove the mouse from the vaporizer and place it on its back on a paper towel and spread the legs.
- Cover the nose of the mouse with a nose cone (a tube connected to the vaporizer that provides a controlled isoflurance dose that comes equipped on some vaporizer units) to maintain anesthetization.
- Gently palpitate the bladder to induce urination and ensure a voided bladder. Wipe the periurethral area with 100% ethanol wipe. Dab the inoculation needle/syringe, point first, into the surgical lubricant.
- Inoculate each mouse with 50 µl of the bacteria solution by inserting the inoculation needle transurethrally, approximately 12 mm, and pressing down on the syringe plunger gently to dispense the inoculum into the bladder gently (10 µl/sec). Remove the inoculation needle from the mouse.
NOTE: Immediate return of inoculum at the urethral opening when beginning to inoculate indicates improper or incomplete insertion of the needle. - Remove the mouse from nose cone and return it to its cage. Repeat steps 3.3-3.8 for each mouse. When/if switching inoculum conditions/strains, dispose of the syringe and inoculation needle in an approved sharps container and start again step 3.2.
NOTE: Inoculation with uropathogenic organisms generally does not cause severe pain symptoms. However, in rare instances, administering large doses of pathogens may cause fever, reduction in food and water intake and abnormal behavior. Animal health should be monitored throughout the experiment. If overt pain symptoms are notice, an analgesic, such as Buprenorphine (0.05−0.1 mg/kg given subcutaneously), can be applied. Procedures should be in accordance with each institution’s IACUC.
