经尿道诱导小鼠尿路感染
Summary
此影片将展示transurethrally诱导小鼠尿路感染及造成的感染程度量化的方法。
Abstract
生长在琼脂利益肾盂肾炎的菌株。一般而言，肾盂肾炎<em> E。大肠杆菌</em>（统一企业）和其他菌株可过夜卢里亚Bertani（LB）琼脂在37 °环境空气中的C。 UPEC菌株的增长为黄白色半透明的菌落，LB琼脂。适当的菌落形态的确认，然后挑单菌落，在肉汤培养。 LB肉汤可用于大多数肾盂肾炎的菌株。可以采用双串口，一夜之间LB培养基培养，以提高I型菌毛，定义为肾盂肾炎细菌致病因子的表达。肉汤培养物稀释至所需浓度的磷酸盐缓冲液（PBS）。 8至12周龄雌性小鼠被放置在异氟醚麻醉和transurethrally用聚乙烯管材覆盖30个计注射器的细菌接种。典型的接种，必须凭经验确定每个细菌/小鼠品系的组合，是10<sup> 6</sup>到10<sup> 8</sup> CFU每鼠标在10至50微升的PBS。 （有一天至数周），尿液样本及膀胱和两个肾脏所需的感染期后收获。各器官剁碎，置于PBS中，并在一个蓝色的子弹匀浆器匀浆。在PBS系列稀释尿液和组织匀浆，并在适当的琼脂培养。翌日，菌落形成单位计数。
Protocol
一，准备尿道导管对于每个鼠标膀胱和肾脏进行采样，5-6不锈钢珠（膀胱，1.6毫米大小0.9-2.0毫米混合）或氧化锆珠（肾，0.5毫米大小），分别放置在一个离心管管。高压灭菌的含珠管。 管冷却至室温后，加入200微升无菌PBS，每个不锈钢含珠管和400微升无菌PBS每个氧化锆珠含管。 我们准备由红，侯德群13所述的尿道导管。高压灭菌一双干净的平头镊子和剪刀（光圈或?…
Discussion
经尿道的细菌接种的小鼠泌尿道感染的感应，是一个历史悠久，但技术要求高的程序11。红和侯德群最近发表的鼠标尿路感染13经尿道前列腺电技术的一个很好的回顾。在本文中，我们试图进一步说明这个实验方法的更细点。
初学者可练习他们的技术，由执行中线剖腹探查术，暴露膀胱，注入了墨汁在PBS（1％）的稀溶液中，观察膀胱蓝色着色。我们发现，一?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者想雪莉谢和珍妮弗佩恩承认他们的专家技术援助。通过小儿泌尿外科学会和斯坦福大学的儿科研究基金的拨款，这项工作成为可能。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 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 Laboratories | 000656 | ||
| Phosphate Buffered Saline (NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO4 1.4mM) | EMD | EM-SX0420-13 (NaCl) EM-PX1405-1 (KCl) EM1.06585.0 (Na2HPO4) EM-5108-1 (KH2PO4) | ||
| LB agar | ||||
| LB broth | EMD | 1.10285.0500 | ||
| Isoflurane (Aerrane) | Baxter | 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 | 212123 | ||
| LB agar powder | Difco | 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 |
Referências
- Schaeffer, A. J., Schwan, W. R., Hultgren, S. J., Duncan, J. L. Relationship of type 1 pilus expression in Escherichia coli to ascending urinary tract infections in mice. Infect Immun. 55, 373-380 (1987).
- Rouschop, K. M. Urothelial CD44 facilitates Escherichia coli infection of the murine urinary tract. J Immunol. 177, 7225-7232 (2006).
- Malaviya, R., Ikeda, T., Abraham, S. N. Contribution of mast cells to bacterial clearance and their proliferation during experimental cystitis induced by type 1 fimbriated E. coli. Immunol LettM. 91, 103-111 (2004).
- Lane, M. C., Alteri, C. J., Smith, S. N., Mobley, H. L. Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract. Proc Natl Acad Sci U S A. 104, 16669-16674 (2007).
- Hvidberg, H. Development of a long-term ascending urinary tract infection mouse model for antibiotic treatment studies. Antimicrob Agents Chemother. 44, 156-163 (2000).
- Hopkins, W. J., Hall, J. A., Conway, B. P., Uehling, D. T. Induction of urinary tract infection by intraurethral inoculation with Escherichia coli: refining the murine model. J Infect Dis. 171, 462-465 (1995).
- Hopkins, W. J., Gendron-Fitzpatrick, A., Balish, E., Uehling, D. T. Time course and host responses to Escherichia coli urinary tract infection in genetically distinct mouse strains. Infect Immun. 66, 2798-2802 (1998).
- Gunther, N. W. t., Lockatell, V., Johnson, D. E., Mobley, H. L. In vivo dynamics of type 1 fimbria regulation in uropathogenic Escherichia coli during experimental urinary tract infection. Infect Immun. 69, 2838-2846 (2001).
- Bishop, B. L. Cyclic AMP-regulated exocytosis of Escherichia coli from infected bladder epithelial cells. Nat Med. 13, 625-630 (2007).
- Thumbikat, P., Waltenbaugh, C., Schaeffer, A. J., Klumpp, D. J. Antigen-specific responses accelerate bacterial clearance in the bladder. J Immunol. 176, 3080-3086 (2006).
- Hagberg, L. Ascending, unobstructed urinary tract infection in mice caused by pyelonephritogenic Escherichia coli of human origin. Infect Immun. 40, 273-283 (1983).
- Hopkins, W. J., Hall, J. A., Conway, B. P., Uehling, D. T. Induction of urinary tract infection by intraurethral inoculation with Escherichia coli: refining the murine model. J Infect Dis. 171, 462-465 (1995).
- Hung, C. S., Dodson, K. W., Hultgren, S. J. A murine model of urinary tract infection. Nat Protoc. 4, 1230-1243 (2009).
Tags
Transurethral InductionMouse Urinary Tract InfectionUropathogenic Bacterial StrainsAgarUropathogenic E. ColiLuria-Bertani AgarColony MorphologyCulture In BrothLB BrothType I PiliVirulence FactorPhosphate Buffered SalineFemale MiceIsoflurane AnesthesiaTransurethral InoculationPolyethylene Tubing-covered SyringesInocula ConcentrationCfu Per MouseUrine SamplesBladderKidneysHarvesting OrgansMinced OrgansHomogenizationBlue Bullet HomogenizerSerial DilutionColony Forming Units
Citar este artigo
Thai, K. H., Thathireddy, A., Hsieh, M. H. Transurethral Induction of Mouse Urinary Tract Infection. J. Vis. Exp. (42), e2070, doi:10.3791/2070 (2010).