建立尿路感染（UTI）模型的能力对于理解细菌致病机制以及推动新型治疗方法的开发至关重要。本研究的目标是展示能够重现并预测人类中观察到的实验结果的小鼠实验性尿路感染模型以及导管相关性尿路感染模型。
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勘误通知
Important: There has been an erratum issued for this article. View Erratum Notice
摘要
摘要
尿路感染（UTI）极为常见，是导致发病率升高的重要原因，且对抗生素治疗的耐药性日益增强。女性患尿路感染的比例明显更高：一生中约有50%的女性会经历至少一次尿路感染。此外，在首次发生尿路感染的女性中，有20%至40%会经历复发，其中部分患者反复发作，严重影响生活质量，伴随疼痛与不适，干扰日常活动，增加医疗成本，且除长期使用抗生素预防外，治疗选择极为有限。尿致病性大肠杆菌（Escherichia coli，UPEC）是社区获得性尿路感染的主要致病菌。导管相关性尿路感染（CAUTI）是最常见的医院获得性感染，每年在美国发生约百万例，造成巨大的医疗负担。尽管UPEC也是CAUTI的主要致病因素，但其他病原体的重要性也逐渐上升，包括粪肠球菌（Enterococcus faecalis）。本文中，我们采用两种已建立的小鼠模型，这些模型可重现人类疾病的多种临床特征。对于尿路感染，C3H/HeN小鼠模型可模拟人类中观察到的UPEC毒力特征，包括宿主免疫反应、胞内细菌群（IBC）的形成以及细菌丝状化现象。对于CAUTI，采用C57BL/6小鼠的模型已被证实对粪肠球菌（E. faecalis）膀胱感染具有易感性，该模型可保留膀胱内的导管植入物。这些代表性模型正在被用于深入揭示尿路感染的发病机制，推动新型治疗方法以及疾病管理与预防策略的开发。
引言
尿路感染（UTIs）是最常见的细菌感染之一，根据感染获得机制可分为两类：社区获得性尿路感染和医院获得性尿路感染。社区获得性尿路感染通常发生在原本健康的女性中，研究表明，约50%的女性在一生中至少会经历一次尿路感染1。此外，复发是一个主要问题。即使接受了适当的抗生素治疗，初次急性感染后的女性在六个月内再次感染的概率仍高达25-40%，且许多女性会持续出现频繁复发2。导致这些感染的细菌对抗生素的耐药性也日益增强，进一步加剧了治疗方案的复杂性3-6。尿路感染每年影响数百万人，在美国每年与医疗相关的费用约为25亿美元，凸显了该疾病的广泛影响和高发病率1,7。医院获得性尿路感染主要与异物存在有关，例如留置导尿管。导管相关性尿路感染（CAUTI）仍是医院获得性尿路感染中最常见的类型，占此类感染的约70-80%8。此外，CAUTI与发病率和死亡率的升高相关，也是继发性血流感染最常见的原因9。
通常认为，与UPEC相关的社区获得性尿路感染是由胃肠道菌群作为储存库，通过性交过程中的机械性操作、卫生不良或其他宿主不同生态位之间的微生物群落动态变化，将细菌引入膀胱所致10。一旦进入膀胱，UPEC会利用多种毒力因子，包括荚膜、铁获取系统、毒素、毒力质粒、tRNA、致病岛以及定植因子，这些因子已被证实参与致病过程11-14。在U....
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方案
伦理声明：所有小鼠感染实验和操作均经华盛顿大学动物研究委员会批准，属于协议编号20150226的一部分，该协议有效期至2018年12月10日。动物的整体护理符合美国国家研究委员会发布的《实验动物护理与使用指南》以及美国农业部动物护理资源指南的要求。安乐死操作符合《美国兽医协会动物安乐死指南（2013年版）》的规定。
1. 尿路致病性大肠杆菌（UPEC）尿路感染实验方案，接种针制备（图S1）
- 取下30 G针头的帽。将约1英寸长的PE10导管套入针杆上。将针组件在紫外线下灭菌过夜。导管针可无限期保存在无菌培养皿中。
2. 尿路致病性大肠杆菌细菌接种物的制备
- 准备UTI89接种物（在接种日前72小时开始）
- 从冻存菌种中将UTI89划线接种至LB（Luria-Bertani）琼脂平板上，于37 °C过夜培养。挑取单菌落，接种于125 ml三角瓶中的10 ml LB液体培养基中，于37 °C静置培养24小时。
- 取10 µl过夜培养物，接种至新的125 ml三角瓶中的10 ml LB液体培养基中，于37 °C继续静置培养24小时。
- 取3 ml培养物（具体体积根据菌株和所需接种量调整）转移至无菌的1.5 ml离心管中，以7,000 × g离心3分钟，弃上清，用1×PBS重悬沉淀，再次离心。将菌体沉淀重悬于1....
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结果
非复杂性和导管相关性尿路感染的膀胱内模型为阐明细菌致病的分子机制、这些疾病对宿主组织的影响，以及开发和测试管理这些常见且高成本感染的新方法提供了灵活的平台。根据小鼠品系和病原体的不同，膀胱内接种可用于研究宿主-病原体相互作用，以阐明启动或调节急性感染所必需的因素图1 和 3），慢性或复发性（图2）膀胱炎。所示数据在 图1 代表典型尿路致病性大肠杆菌（UPEC）膀胱炎分离株UTI89在急性期（24 hpi）对尿路的定植情况 44接种后，让感染在小鼠体内发展24小时，随后处死小鼠并匀浆膀胱和肾脏组织。将组织匀浆液进行系列稀释后涂布平板，以计数定植细菌。在某些小鼠品系（尤其是C3H/HeN）中，可建立并维持慢性尿路致病性大肠杆菌（UPEC）定植及膀胱炎症，其建立依赖于感染剂量和尿路致病菌的种类。慢性膀胱炎定义为持续性高滴度菌尿（>104 CFU/ml）、膀胱炎症及高滴度细菌性膀胱负荷（>104 CFU/膀胱）在处死时 33.......
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讨论
单纯性社区获得性尿路感染是一种常见且治疗成本较高的感染，每年导致数百万例初级医疗就诊46。此外，导管相关性尿路感染（CAUTI）是一种常见的医疗机构获得性感染，由于美国医疗保险和医疗补助服务中心不再为因医院内获得性CAUTI所增加的治疗费用向医疗机构提供补偿，因此给医疗服务系统带来了巨大的经济负担45。本方案中描述的尿路感染小鼠模型，包括单纯性膀胱炎和导管相关性尿路感染模型，为深入理解宿主与病原体之间在启动、维持及调控多种类型尿路感染过程中所必需的分子机制提供了不可替代的研究工具。这些模型使得研究人员能够应用强大的细菌遗传学和小鼠遗传学、免疫学、显微成像、生物化学以及化学生物学等技术手段对尿路感染进行系统分析23,47-49。此外，本文所述的实验方案还为开发和测试新型治疗及预防策略提供了平台，包括细菌毒力的小分子抑制剂、以及新的疫苗靶点和免疫策略50-55。
成功应用单纯性膀胱炎模型必须维持若干关键条件。与所有小鼠麻醉操作一样，需谨慎操作以防止因麻醉过量导致小鼠死亡。在制备接种导管时，硅胶管的长.......
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披露
作者声明不存在任何竞争性经济利益。
致谢
本工作的资助来自 ORWH SCOR P50 DK064540、RO1 DK 051406、RO1 AI 108749-01、F32 DK 101171 和 F32 DK 104516-01。
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材料
| 姓名 | 公司 | 目录编号 | 评论 |
|---|---|---|---|
| 导管和针头制备材料： | |||
| 30 G 针头 | BD Precision Glide | 305106 | 30 G x ½ (0.3 mm x 13 mm) |
| PE10 聚乙烯导管 | BD | 427400 | 内径 -0.011 英寸 (0.28 mm)；外径 – 0.024 英寸 (0.61 mm) |
| RenaSIL 025 铂固化硅胶导管 | Braintree Scientific, Inc | SIL 025 | 内径 0.012 英寸 × 外径 0.025 英寸，25 英尺线圈 |
| 感染实验材料： | |||
| 异氟烷 – Isothesia | Butler Schein | 29405 | 250 ml |
| 透明直壁玻璃罐 | Kimble Chase | 5413289V 21 | |
| 不锈钢茶叶过滤器 | Schefs-Amazon | Schefs 品牌优质散装茶叶过滤器 - 不锈钢 - 大容量 - | |
| 非无菌棉球 | Fisherbrand | 22-456-880 | |
| 50 ml Falcon 管 | VWR | 89039-660 | |
| Isotec 3 - 蒸发器 | Ohmeda | 1224478 | |
| 耳标打孔器 | Fisher Scientific | 13-812-201 | （必要时使用） |
| 聚维酮碘溶液 | Betadine solution | 10% 聚维酮碘外用溶液 | |
| 棉签 | Fisher Scientific | 22-037-924 | 6 英寸 |
| 实验台用垫巾 | Fisherbrand | 14206 63 | 吸水垫（20” × 36” 垫子） |
| 外科润滑剂 | Surgilube | 0281-0205-36 | |
| 解剖剪刀 | Fine Science tools, INC | 14084-08 | |
| 微型 Adson 镊子 | Fine Science tools, INC | 11018-12 | |
| 1 ml 注射器 | BD | 309659 | 结核菌素滑动接头 |
| Parafilm 封口膜 | Bemis | PM996 | 4 英寸 × 125 英尺 |
| Eppendorf 管架 | Fisherbrand | 05-541-1 | |
| Eppendorf 管 | MIDSCI | AVX-T-17-C | |
| 导管、膀胱和肾脏采集材料： | |||
| 匀浆器 | PRO Scientific INC | Bio-Gen Pro 200 | |
| 5 ml 聚丙烯圆底管 | BD | 352063 | 用于器官匀浆 |
| 纸巾 | Georgia-Pacific | ||
| 乙醇 | Pharmco-AAPER | 11100020S | 200 proof |
| Costar™ 透明聚苯乙烯 96 孔板 | Corning | 3788 | |
| 1× 磷酸钠盐缓冲液 | Sigma-Aldrich | P3813 | |
| BRANSONIC 超声波清洗机 1210 | Branson Ultrasonics Corporation | 1210 | |
| IBC 实验材料： | |||
| 6 孔组织培养板 | Techno Plastic Products | 92006 | |
| 固定针 | Fine Science Tools | 26002-20 | |
| Sylgard 184 | Dow Corning | 3097358-1004 | 硅橡胶试剂盒 |
| X-gal（5-溴-4-氯-3-吲哚基-β-D-半乳糖苷） | Invitrogen | 15520-034 | 超纯 |
| N,N-二甲基甲酰胺 | Sigma Aldrich | D4551 | |
| MgCl2（氯化镁） | Sigma Aldrich | M8266 | |
| 脱氧胆酸钠 | Sigma Aldrich | D6750 | |
| Nonidet-P40 | Roche | 11754599001 | 辛基酚聚乙二醇醚 |
| 六氰合铁(II)酸钾三水合物（亚铁氰化钾） | Sigma Aldrich | P3289 | |
| 六氰合铁(III)酸钾（铁氰化钾） | Sigma Aldrich | 60299 |
参考文献
- 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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重印与许可
勘误
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.
