作者感谢林恩·福斯特在感染实验中的技术援助，华盛顿大学细胞成像中心（WUCCI）的詹姆斯·菲茨帕特里克访问SEM，斯科特·胡尔特格伦负责UTI89kanR UPEC菌株，大卫·亨斯塔德对手稿进行了批判性阅读。
这项工作得到了美国国家科学基金会（VPO#DGE - 1143954研究生研究奖学金），华盛顿大学医学院妇女传染病研究中心（NMG试点研究奖），美国心脏协会：#12POST12050583（NMG）和#14POST20020011（NMG）以及美国国立卫生研究院NIAID：R01 AI114635（ALL）和NIDDK的支持： R21 DK092586 （ALL）、P50 DK064540-11 （SJH， 项目 II PI：ALL） 和 K01 DK110225-01A1 （NMG）。一些动物研究是在NCRR拨款C06 RR015502支持的设施中进行的。华盛顿大学细胞成像中心（WUCCI，SEM在那里进行）和MSJ得到了华盛顿大学医学院，华盛顿大学儿童发现研究所和圣路易斯儿童医院（CDI-CORE-2015-505），巴恩斯犹太医院基金会（3770）和国家神经疾病和中风研究所（NS086741）的支持。资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。

作者声明他们没有与本研究相关的利益冲突。

该模型中的第一个关键步骤是鉴定在原发性UTI阶段尚未清除UPEC菌尿的小鼠。这些小鼠必须从实验中取出，否则它们会混淆 阴道G. 暴露后UPEC菌尿的发生率。初始 UPEC 接种后，应每周收集尿液以监测细菌清除率。大约65-80%的C57BL / 6小鼠将在4周内清除UTI89kanR 感染。其他自交小鼠品系对UPEC间隙42，43 和储层形成具有不同的倾向，因此可能不适合该模型。第二个关键点是，实证研究已经确定， 阴道毛滴虫 的两次连续接种（间隔12小时或1周）对于触发仅在暴露于PBS的对照小鼠中发生的背景自发出现之上的显着储库出现是必要的。两个连续暴露之间的其他持续时间尚未测试，但可能会产生类似的结果。重要的是要注意，仅在 阴道毛孢 杆菌暴露1周的模型中观察到UPEC膀胱滴度的降低39。虽然可以进行两次以上的暴露，但经验证据表明，仅重复导尿就会增加出苗，这可能会混淆对结果的解释，或者需要更多的动物来区分暴露组和对照组之间的差异。最后，原位膀胱固定方法有几个关键步骤。需要一些技能来确保固定剂保持在夹紧的膀胱内。放气的膀胱将更难通过SEM成像。将固定剂接种到膀胱中时，非常温和也很重要，因为用含固定剂的导管刮擦尿路上皮可以诱导尿路上皮去角质，而与 阴道球菌引发的是什么无关。固定剂鸡尾酒中提到的所有浓度都是最终浓度。这些比例不当会导致细胞的固定和肿胀或收缩不足。固定剂应加热至生理温度，以避免细胞和组织的温度冲击。变暖还略微改善了固定剂通过质膜的扩散速率。虽然对于制备用于SEM分析的样品，通常可以省略锇染色，但这是该方案中稳定脂质并防止细胞膜在临界点干燥期间开裂的重要步骤。
可以修改该协议以分别测试其他UPEC和/或 阴道芽孢杆菌 菌株形成储库和触发其出现的能力。还可以添加其它实验因素，例如暴露于其它阴道细菌（例如， 透明乳杆菌 PVAS100）或热杀伤 的阴道G.，这两者在该模型39中均未表现出病理学。在选择其他细菌菌株进行测试时，重要的是要证明生长一致，以便在所有实验中使用标准接种物浓度。JCP8151BSmR 的生长已在厌氧室中进行了优化。该菌株可能在厌氧GasPak系统中培养，但这需要优化以确保强大的细菌生长。最后，可以修改模型中某些步骤的时间。例如，可以在UPEC储层形成阶段的早期时间点收集尿液，以监测CFU或宿主反应。在该模型中未观察到在早期时间点（3，6，12 hpi）收集尿液样本对感染进展或建立储库的不利影响。据报道，UPEC储层的出现发生在给予两次JCP8151BSmR 剂量12小时或1周后，但其他时间间隔尚未测试。也可以通过将UPEC储库形成阶段减少到2周（而不是4周）来减少模型的总时间长度，因为许多小鼠此时已经清除了菌尿。先前检查膀胱暴露于化学去角质剂后UPEC出现的研究使用了1或2周的UPEC储层形成阶段17，18。然而，减少UPEC菌尿清除的时间可能是以需要从实验中剔除更多动物为代价的。最后，可以在其他时间点进行膀胱的SEM分析，以观察 阴道G. 对尿路上皮的影响的持续时间。
关于故障排除，有一些重要的考虑因素，特别是关于膀胱SEM分析。根据所使用的小鼠背景和炎症的程度，一些膀胱会出现非常薄的壁。这些膀胱在临界点干燥期间往往会卷曲更多，并可能导致类似壳的形状。如果发生这种情况，最好的方法是沿着卷曲的界面将壳状膀胱切成两半，然后第二次去除大部分悬垂组织。使用PTFE涂层的双刃剃须刀片进行切割效果最佳。多余的脂肪有时会在锇染色步骤中溶解。这可能导致不需要的不溶性脂肪飞沫，在冲洗和脱水步骤中可能无法洗掉，并且在随后的干燥过程中可能会沉降在膀胱表面。这些液滴可以表现为散布在样品上的小球体或圆盘状结构（图4D）。这可以通过确保从膀胱周围去除尽可能多的脂肪组织来缓解。铂可以代替铱涂层，但厚度应保持在最低限度，以减少对精细结构细节的遮蔽。强烈建议在涂装过程中使用旋转平台。
该模型的一个局限性是它需要大量的小鼠。只有65-80%的C57BL / 6小鼠会清除其UPEC菌尿，并适合随后的 阴道G. 或PBS接种（见 图2C）。为了每组获得10-12只小鼠（阴道接种G. 与PBS），应首先感染约30只小鼠UPEC。此外，可能需要多次实验才能实现检测统计显著性所需的生物学重复。当暴露间隔1周时，UPEC出现在暴露于PBS的14%的小鼠中（图3B）。因此，检测 阴道假单胞菌 暴露小鼠中相对于PBS对照组（功率为0.8; α = 0.05 [单侧]）的UPEC rUTI显着增加需要测试每个暴露组的累积总数至少40只小鼠。另一个考虑因素是，这些实验是昂贵和劳动密集型的。必须每周监测小鼠的UPEC清除率，实验时间过程为4-5周，具体取决于 阴道G. 是在12小时的时间内给予两次还是间隔两次1周。SEM是劳动密集型的，并且可能很昂贵，具体取决于显微镜的可用性和服务费。为SEM准备整个气囊为分析提供了丰富的材料，但缺点是分析每个气囊可能非常耗时。因此，与用于尿液和组织滴度的较高动物数量相比，SEM可能只能分析有限数量的膀胱。此外，获得膀胱“杯子”弯曲表面的高质量图像需要技能，因为阴影会阻碍能见度。虽然膀胱扫描电镜检查是可视化尿路上皮去角质的有用工具，但这种方法在很大程度上是定性的。由于样品固定在圆形，并且由于在固定剂中使用戊二醛，因此无法通过光学显微镜筛选荧光表达的细菌。免疫染色和化学染料与该过程不相容，因为使用戊二醛会交联大多数抗原和锇，并且会掩盖抗原位点并使组织变暗。也就是说，SEM技术对于无需使用其他探针即可定量评估的参数非常有用，例如细胞大小48，49。
此模型提供了除前面描述的方法之外的几个优点。它允许检查由膀胱储液器出现引起的UPEC rUTI的机制，而不是从外部来源重新引入膀胱。由于膀胱储液器出现的rUTI的其他模型使用化学试剂（硫酸鱼精蛋白或壳聚糖）引起尿路上皮去角质17，18，这不会是女性rUTI的触发因素。 阴道假单胞菌 是一种流行的泌尿生殖系统细菌，在一些女性中，通过导管插入术或耻骨弓上抽吸直接从膀胱收集的尿液中检测到23，26.这一事实，加上BV（ 阴道毛果在阴道 中过度生长）与UTI之间的已知关联，表明 阴道毛滴虫 是rUTI的临床上合理的触发因素。最后，原位膀胱固定方法保留了膀胱超微结构并限制了损伤，确保膀胱层不会彼此分离。以前用于可视化尿路上皮的方法传统上是让用户无菌地收获，一分为二，拉伸并将膀胱固定在解剖盘上，然后将拉伸的膀胱浸入固定剂48中。这种方法产生非常平坦的样品，但不能确保组织的均匀或自然拉伸，并且可能导致过度和不足的区域拉伸（导致高度褶皱的组织），并可能导致膀胱层分离。此外，这些对膀胱的物理操作以拉伸和固定组织会导致损伤，包括尿路上皮去角质。另一种方法是将完整的膀胱浸没在固定剂中，然后包埋在石蜡中并用切片机获取薄片。薄切片对于检查细菌和宿主蛋白定位的免疫组织化学实验非常有价值，但薄切片不允许尿路上皮表面的可视化。这种SEM方法允许一次检查整个膀胱的表面。
如前所述，该模型的未来应用包括测试其他UPEC菌株以确定它们是否形成细胞内储库，并测试其他 阴道芽孢杆菌 菌株以评估它们是否引起角质和UPEC出现导致rUTI。C57BL / 6小鼠以外的其他小鼠菌株也可以进行测试，尽管不建议使用具有高发展慢性膀胱炎倾向的小鼠（例如C3H背景上的小鼠），因为需要从实验中剔除太多的小鼠。C57BL / 6小鼠的另一个优点是许多基因敲除菌株是市售的。这种菌株为询问储层形成和/或出现所涉及的宿主因素提供了机会。

尿路感染（UTI）在全球范围内带来了巨大的医疗负担，每年影响数百万人的生活质量，尤其是女性1。尿路致病性大肠杆菌 （UPEC） 是 UTI1 的最常见病因。许多发生 UTI 的患者（约 20-30%）会在 6 个月内出现复发性 UTI （rUTI），尽管抗生素介导的初始感染已清除2。不幸的是，多达5%的绝经前妇女每年患有3次或更多的rUTI3，4。rUTI的序贯发作可能是由指示病例5，6，7，8中相同的UPEC菌株的持续存在引起的。来自人类样本和小鼠模型的数据表明，同菌株rUTI可能是由UPEC居住在膀胱的静止储层中引起的。在人类中，在UTI9，10，11，12，13患者的上皮细胞和膀胱活检中检测到UPEC。C57BL / 6小鼠的研究表明，一些UPEC菌株可以在膀胱中建立静止的细胞内储库，通过荧光显微镜和膀胱组织的匀浆和培养检测到，这些储库在菌尿14，15，16消退后维持数月。用诱导膀胱上皮剥落的药物（尿路上皮）治疗膀胱，例如硫酸鱼精蛋白17或壳聚糖18，触发UPEC从储库出现引起rUTI。这些数据表明，在既往感染中携带膀胱 UPEC 储液器的女性中，导致尿路上皮去角质的膀胱暴露可能引发 rUTI。
越来越多的证据表明，阴道微生物群会导致尿路感染19，20。阴道加德纳菌是阴道和泌尿微生物群21，22，23，24，25，26，27，28，29的频繁成员。在阴道中，高水平的阴道G.的存在与称为细菌性阴道病（BV）的微生物生态失调有关，其影响约30%的女性30，31，32。与乳酸杆菌33，34，35，36，37的阴道社区的女性相比，BV女性患UTI的风险更高。在小鼠模型中，阴道G.在阴道38和膀胱39中引起上皮去角质。在携带 UPEC 膀胱储液器的 C57BL/6 小鼠中，两次连续膀胱暴露于阴道 G. 但未暴露于 PBS - 导致 UPEC 从储集层中重新出现，导致 UPEC rUTI。与PBS暴露的对照动物相比，先前已解决UPEC菌尿的小鼠尿液中UPEC滴度的出现以及随后UPEC膀胱匀浆滴度的降低证明了这一点。有趣的是，膀胱中的阴道G.没有持久的定植。在绝大多数情况下，两次短暂暴露，每次尿液中存活的阴道 G. 
该协议描述了由居住在细胞内膀胱储液器中的UPEC引起的rUTI小鼠模型，使用 阴道G. 膀胱接种来触发复发。该模型取得的进步是，与以前使用的化学试剂相比， 阴道G. 是rUTI的临床相关生物触发因素。此外，小鼠尿路阴道 球菌 的相对较短的存活率允许检查短暂微生物暴露对尿路上皮的影响，如性活动后可能发生的那样。除了概述rUTI模型外，该协议还描述了尿液细胞学和原位膀胱固定以及通过扫描电子显微镜（SEM）对尿路上皮进行成像的方法。
阴道芽孢杆菌诱导的复发性UPEC UTI的该方案使用带有卡那霉素抗性盒（UTI89kanR）的UPEC菌株UTI89 40。并非所有测试的UPEC菌株都能够在小鼠41的急性感染阶段形成细胞内细菌群落，目前尚不清楚UPEC的所有菌株是否都具有形成潜伏细胞内储库的能力。在模型中使用其他UPEC菌株之前，应确认储层的形成。该方案使用自发性链霉素耐药阴道G.分离株JCP8151BSmR38。通过 JCP8151BSmR 诱导 rUTI 需要两次连续阴道 G. 接种，间隔 12 小时或 7 天 （d）39。其他阴道芽孢杆菌菌株是否诱发去角质和/或UPEC rUTI仍有待该模型确定。必须使用具有已知抗生素耐药性的 UPEC 和阴道菌芽孢杆菌菌株（例如用于 UPEC 的卡那霉素或大观霉素以及用于阴道菌的链霉素），因为可以将抗生素添加到琼脂平板上，以防止内源性小鼠微生物群的生长，否则这些微生物群可能会干扰列举集落形成单位 （CFU） 以监测感染。这对于培养尿液标本尤其重要，因为小鼠尿液中经常含有其他细菌，这些细菌可以在没有抗生素的情况下在培养板上过度生长。这些内源性细菌在小鼠尿液中的起源尚不清楚，但可能反映了尿液收集过程中发现的尿道周围和泌尿生殖系统细菌。
阴道假单胞菌是一种兼性厌氧菌，因此，该方案描述了在厌氧室中生长的阴道菌JCP8151BSmR。如果没有厌氧室，可以使用其他方法来维持厌氧生长条件（例如气密容器中的GasPak袋）。或者，阴道芽孢杆菌的一些菌株（包括JCP8151BSmR）将在标准组织培养箱（5%CO2）中生长。正如使用除 JCP8151BSmR 以外的阴道 G. 菌株需要进行测试以确保细菌在该模型中的行为相似一样，改变生长条件需要经验性地确定培养的理想持续时间（在平板上和液体中）和光密度 （OD）600 当量，以达到所需的可行接种物浓度。此外，尚不清楚生长条件是否影响阴道 G.
最后，在考虑是否利用该模型时，研究人员应该意识到，与典型的UTI小鼠模型相比，每组可能需要更多的动物。这部分是因为诱导rUTI需要小鼠解决由膀胱初始感染引起的UPEC菌尿。因此，任何未能清除菌尿（通常表明持续肾脏感染的表型）的小鼠均不包括在方案的rUTI阶段。为这些研究提供动力所需的小鼠数量也受到“自发”UPEC进入尿液的速率（平均12-14%）的影响。最后，不同的小鼠品系与细胞内储库形成42，43具有不同的发生慢性菌尿的倾向。如果在此模型中使用C57BL / 6以外的小鼠菌株，则必须确认动物发展出静止的UPEC细胞内储库。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>30G x 1/2 needles</td>
			<td>BD</td>
			<td>305106</td>
			<td>for catheters</td>
		</tr>
		<tr>
			<td>5 1/2&#34; straight forcep hemostat</td>
			<td>McKesson</td>
			<td>487377</td>
			<td>in situ bladder fixation</td>
		</tr>
		<tr>
			<td>ACE 600 Sputter coater</td>
			<td>Leica</td>
			<td></td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>aluminum SEM stub</td>
			<td>Ted Pella</td>
			<td>16111</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>EMS</td>
			<td>12340</td>
			<td>in situ bladder fixation</td>
		</tr>
		<tr>
			<td>conductive carbon adhesive tab&#160;</td>
			<td>Ted Pella</td>
			<td>16084-1</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>Conductive silver paint</td>
			<td>Ted Pella</td>
			<td>16034</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>CPD 300 Critical Point Drier</td>
			<td>Leica</td>
			<td></td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>Cytofunnel metal clip</td>
			<td>Simport</td>
			<td>M964B</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMS</td>
			<td>15050</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td>for NYCIII G. vaginalis growth media</td>
		</tr>
		<tr>
			<td>glutaraldehyde</td>
			<td>EMS</td>
			<td>16320</td>
			<td>in situ bladder fixation</td>
		</tr>
		<tr>
			<td>Hema 3 staining kit</td>
			<td>Fisher</td>
			<td>23123869</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Cellgro&#160;</td>
			<td>25-060-Cl</td>
			<td>for NYCIII G. vaginalis growth media</td>
		</tr>
		<tr>
			<td>iridium</td>
			<td>Ted Pella</td>
			<td>91120</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>isofluorane</td>
			<td></td>
			<td></td>
			<td>mouse anaesthesia</td>
		</tr>
		<tr>
			<td>kanamycin</td>
			<td>Gibco</td>
			<td>11815024</td>
			<td>add to UPEC LB selective plates (50 ug/mL)</td>
		</tr>
		<tr>
			<td>Luria-Bertani agar</td>
			<td>BD</td>
			<td>DF0445174</td>
			<td>UPEC growth plates</td>
		</tr>
		<tr>
			<td>Luria-Bertani broth</td>
			<td>BD</td>
			<td>DF0446173</td>
			<td>UPEC growth media</td>
		</tr>
		<tr>
			<td>Merlin FE-SEM</td>
			<td>Zeiss</td>
			<td></td>
			<td>scanning electron microscope</td>
		</tr>
		<tr>
			<td>Milli-Q Water Purifier</td>
			<td>Millipore</td>
			<td>IQ-7000</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>for NYCIII G. vaginalis growth media</td>
		</tr>
		<tr>
			<td>Olympus Vanox AHBT3 microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>osmium tetroxide</td>
			<td>EMS</td>
			<td>19170</td>
			<td>SEM sample processing</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>EMS</td>
			<td>15710</td>
			<td>in situ bladder fixation</td>
		</tr>
		<tr>
			<td>polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427401</td>
			<td>for catheters</td>
		</tr>
		<tr>
			<td>Proteose Peptone #3&#160;</td>
			<td>Fisher&#160;</td>
			<td>DF-122-17-4</td>
			<td>for NYCIII G. vaginalis growth media</td>
		</tr>
		<tr>
			<td>PTFE coated double edge razor blade</td>
			<td>EMS</td>
			<td>72000</td>
			<td>cutting bladders for SEM</td>
		</tr>
		<tr>
			<td>Shandon Cytocentrifuge</td>
			<td>Thermo Scientific</td>
			<td>A78300002</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>Shandon cytofunnel filter</td>
			<td>Simport</td>
			<td>M965FWDV</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>Shandon Double cytofunnel</td>
			<td>Simport</td>
			<td>M964-1D</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>Shandon double cytoslides (coated)</td>
			<td>Thermo Scientific</td>
			<td>5991055</td>
			<td>cytospun urinalysis</td>
		</tr>
		<tr>
			<td>sodium cacodylate trihydrate</td>
			<td>EMS</td>
			<td>12310</td>
			<td>in situ bladder fixation</td>
		</tr>
		<tr>
			<td>spectrophotometer</td>
			<td>BioChrom</td>
			<td>80-3000-45</td>
			<td>measuring bacterial OD600</td>
		</tr>
		<tr>
			<td>streptomycin</td>
			<td>Gibco</td>
			<td>11860038</td>
			<td>add to G. vaginalis NYCIII selective plates (1 mg/mL)</td>
		</tr>
		<tr>
			<td>tuberculin slip tip syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td>for catheters</td>
		</tr>
		<tr>
			<td>Yeast Extract&#160;</td>
			<td>Fisher</td>
			<td>DF0127-17-9</td>
			<td>for NYCIII G. vaginalis growth media</td>
		</tr>
	</tbody>
</table>

recurrent escherichia coli urinary tract infection triggered by gardnerella vaginalis bladder exposure in mice

由泌尿致病性 大肠杆菌 （UPEC） 引起的复发性尿路感染 （rUTI） 很常见且费用高昂。以前描述雄性和雌性小鼠UTI模型的文章已经说明了尿液和组织中的细菌接种和计数程序。在C57BL / 6小鼠的初始膀胱感染期间，UPEC在膀胱上皮细胞内建立潜伏储库，这些储库在UPEC菌尿清除后持续存在。该模型建立在这些研究的基础上，以检查由潜伏膀胱储层内出现的UPEC引起的rUTI。在该模型中，泌尿生殖器细菌 加德纳菌 阴道被用作rUTI的触发因素，因为它经常存在于女性的泌尿生殖道中，特别是在与UTI相关的阴道生态失调的情况下。此外，还描述了一种原位膀胱固定然后对膀胱组织进行扫描电子显微镜（SEM）分析的方法，并有可能应用于涉及膀胱的其他研究。

接种后，尿液中可检测到UPEC滴度（图2B）。未能在含有卡那霉素的选择性培养基上接种尿液样本可能会导致污染尿液的内源性小鼠微生物群过度生长。UPEC菌尿水平在第1天可能很高，并且可能在第一周内增加，然后在稍后的时间点降低（图2C）。大约65-80%的小鼠在28 dpi的尿液中没有可检测到的UPEC（图2C，绿色圆圈）。这些鼠标可以在模型的后续步骤中使用。保持细菌的小鼠（图2C，红色椭圆）应从实验中消除。
阴道 G. 的 两次连续暴露间隔 12 小时（图 3A）或间隔 1 周（图 3B）导致细胞内储库出现 UPEC，导致复发性菌尿。与PBS对照组相比，暴露于 阴道G 的小鼠中UPEC菌尿水平（曼惠特尼试验）和显示UPEC rUTI的小鼠比例（费舍尔精确测试）显着更高。尿液细胞学分析检测来自阴道暴露的 G.小鼠尿液中的PMN，这些小鼠显示UPEC出现（图3C）。在间隔1周的两次暴露的模型中，与PBS相比，阴道暴露的 G.小鼠膀胱组织中的UPEC滴度较低（图3D），可能是由于UPEC从储库和随后的清除中出现。
通过SEM对原位固定膀胱组织的可视化显示，仅暴露于PBS的对照小鼠的膀胱表面排列的大浅表伞形尿路上皮细胞（图4A）。尿路上皮去角质的证据是浅表伞状细胞的丧失，揭示了暴露于 阴道芽孢杆菌 的小鼠中较小的潜在下移行上皮细胞（图4B）。在UPEC接种后的早期，在细胞内储库建立期间，UPEC在尿路上可见，并且从去角质细胞中长出细丝（图4C）。

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61967/61967fig02.jpg"> 图 2.在第1阶段（储库形成）期间监测尿液中的UPEC滴度。 （A） 菌落形成单元（CFU）电镀示意图。（B）尿中UPEC滴度的LB + 卡纳霉素的代表性图像。黑色圆圈表示尿液样本斑点，应进行计数以计算 CFU/mL。（C）C57BL / 6小鼠中细菌尿的时间过程。每条线代表一只小鼠，追踪UPEC尿液滴度随时间的变化。虚线表示检测限 （1000 CFU/mL）。红色椭圆表示四只小鼠（共20只）未能解决UPEC菌尿，因此不会用于 阴道G.诱导的rUTI模型。相反，绿色圆圈表示小鼠解决了UPEC菌尿并进入后续阶段。（D） 用于生成图的数据表 C. 黄色、可检测的CFU;绿色，无丙氧丙酸。（E）根据尿液中不再检测到UPEC CFU的时间点将小鼠随机分配到暴露组（“解决日”）。面板 D 左列中的鼠标编号与面板 E 中给出的鼠标编号相同， <a href="https://www.jove.com/files/ftp_upload/61967/61967fig02large.jpg" target="_blank">请单击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61967/61967fig03.jpg"> 图 3. 阴道 G. 触发 UPEC 鲁蒂。 UPEC在两次连续尿路暴露于PBS（圆圈）或 阴道G .（格宏体;方块）后，尿液中的滴度为12小时（A）或1周（B）。每个符号代表一个单独的鼠标。绘制第二次暴露后1-3天之间从每只小鼠检测到的最高CFU / mL UPEC。在没有可检测菌尿的小鼠绘制在检测极限处（虚线）。（C）尿液细胞学分析显示UPEC（箭头）和多形核（PMN）细胞（箭头）。比例尺 = 20 μm。（D）膀胱组织中的UPEC滴度在两次连续尿路暴露后收集3 d，间隔1周。每个符号代表不同的鼠标，零在检测极限处绘制（虚线）。在 A、B 和 D 中，箱子位于第一和第三个四分位数处，其中中位数标记，晶须从最小到 最大 <。 ** P < 0.01; P < 0.0001。 <a href="https://www.jove.com/files/ftp_upload/61967/61967fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61967/61967fig04.jpg"> 图 4.原位固定膀胱的扫描电镜分析。 在两次暴露于PBS（A）或 阴道G （C）后3小时（间隔12小时）从小鼠收集膀胱。虚线表示单个尿上皮细胞，该细胞在阴道暴露的 膀胱中较小，因为大的浅表细胞已经脱落，露出潜在的过渡上皮。（B）在模型第1阶段首次接种UPEC后6小时收集膀胱，显示尿路上皮去角质和细胞外UPEC。（D）膀胱表面存在的不溶性脂肪滴的例子。比例尺在主图像中为20 μm，在插图中为2 μm。 <a href="https://www.jove.com/files/ftp_upload/61967/61967fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice at JoVE.com

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

展示了产尿道 大肠杆菌 （UPEC） 经尿道接种以建立潜伏细胞内膀胱储液囊的小鼠模型，随后膀胱暴露于 阴道 G. 以诱导复发性 UPEC UTI。还证明了细菌的计数，尿液细胞学检查以及原位膀胱固定和扫描电子显微镜处理。

由加德纳菌阴道膀胱暴露在小鼠中引发的复发性大肠杆菌尿路感染

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

recurrent-escherichia-coli-urinary-tract-infection-triggered

Research

JoVE Journal

Immunology and Infection

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

5.7K 次观看.  Washington University School of Medicine in Saint Louis. 展示了产尿道 大肠杆菌 （UPEC） 经尿道接种以建立潜伏细胞内膀胱储液囊的小鼠模型，随后膀胱暴露于 阴道 G. 以诱导复发性 UPEC UTI。还证明了细菌的计数，尿液细胞学检查以及原位膀胱固定和扫描电子显微镜处理。

视频: Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Transurethral Induction of Mouse Urinary Tract Infection

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&deg;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.

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

经尿道诱导小鼠尿路感染

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

科研

免疫与感染

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

复发性单纯疱疹性角膜基质性角膜炎小鼠模型研究人类HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

一步法负色谱纯化<em&gt;幽门螺杆菌</em&gt;中性粒细胞激活蛋白过度表达<em&gt;大肠杆菌</em&gt;在批处理模式

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

小动物模型尿路感染的实验研究: 男性和女性的经尿道插管

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

在大肠杆菌中生成特异的乙酰蛋白的简易协议

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.