我们从医学研究部、美梅医疗中心 (台湾台南) 等地认识到, 帮助小鼠感染, 以及国立诚武大学实验动物中心的支持。这项工作得到了科技部长 (多数) 赠款 (多数 104-2321-006-019、105-2321-b-006-011、and106-2321-B-006-005) 的支持。

注意: 根据疾病控制和预防中心 (CDC) 生物安全指导 (https://www.cdc.gov/), 大肠杆菌 O157:H7 是生物安全等级 2 (BSL-2) 病原体。因此, 所有涉及肠出血性大肠杆菌的实验程序都必须在 BSL-2 设施中进行。在进行实验时, 要穿上实验室的大衣和手套。在认证的生物安全柜 (BSC) 工作。对实验台进行70% 乙醇消毒实验。所有接触 (或潜在接触) 肠出血性大肠杆菌的仪器或设备应使用70% 乙醇或漂白剂进行消毒。污染 (或可能污染的) 废物应小心密封和蒸压。如有必要, 戴上口罩、护眼、双手套或连身裤。6周大的 C57BL/6 雌性小鼠在国立成武大学实验动物中心 (NCKU) 购买和维护。动物实验由 NCKU 的机构动物护理和使用委员会批准 (批准号 104-039)。
1. Bioluciferase 标记的大肠杆菌生成
<ol><li>混合50的脱氧核糖核酸 (脱氧核糖核酸), 1 ul 每10微米向前和反向底漆, 2 ul 2.5 毫米 deoxynucleotides (dNTPs), 2 ul 10x 缓冲器, 0.2 ul DNA 聚合酶, 和无菌的双蒸馏水 (ddH2O) 到最后一卷 20 ul, 以放大抗卡那霉素卡带, nptII基因24。DNA 模板来自 pBSL18024, 它是从国家 BioResource 项目 (NBRP) 购买的。PCR 条件列在表 1中, 并且底漆序列在表 2中可用。</li>
	<li>将 PCR 产品结扎在套件协议之后的商业克隆载体上 (请参见材料表)。</li>
	<li>将nptII基因片段从克隆向量中的NsiI和SmaI和克隆复制到 pBBR1MCS4 中, 由NsiI和ScaI从 pAKlux29中消化, 以创建卡那霉素抗性质粒,pWF27813。</li>
	<li>将luxCDABE操纵从表达质粒9、pAKlux2、 SpeIhf和ScaI的荧光素酶和克隆到SpeI-hf和SmaI中消化 pWF278 生成卡那霉素抗性和荧光素酶表达质粒, pWF27913。 注: 对于质粒提取物和切除片段纯化, 分别采用商业质粒萃取和凝胶萃取试剂盒。为酵素消化, 混合 100 ng 脱氧核糖核酸, 1 ul 10x 缓冲器, 1 ul 每选择的制约酵素和无菌 ddH2O 到总容量 10 ul, 并且孵化在37°c 为 2.5-3 h。</li>
	<li>将 pWF279 质粒转化为大肠杆菌 O157:H7 EDL933 主管细胞, 用2500伏电穿孔4毫秒。</li>
	<li>将转化后的细菌细胞在1毫升 Luria Bertani (LB) 培养基中孵育1小时, 37 摄氏度。</li>
	<li>板上的细菌在 LB 琼脂板块补充50µg/毫升卡那霉素在37摄氏度, 16-18 小时。</li>
	<li>第二天, 通过体内成像机检查板的发光信号。选择一个单一的殖民地从板块和文化它在3毫升 LB 补充50µg/毫升卡那霉素在37°c 16-18 h。</li>
	<li>在无菌 ddH2O 中稀释100% 甘油, 制备细菌培养基, 使30% 甘油溶液。</li>
	<li>冷冻卡那霉素耐药性的大肠杆菌O157:H7 EDL933 细菌, 窝藏荧光素酶质粒作为一个细菌库存的螺帽 cryovial 作为1:1 的细菌培养率和30% 甘油溶液-80 摄氏度。甘油的最后浓度是15%。</li>
</ol>2. 生物发光大肠杆菌制备口服疫苗
注: 在图 1中介绍了大肠杆菌制备和小鼠口服饲实验程序的时间流程图, 以帮助实验性的准备工作。
<ol><li>条纹大肠杆菌O157:H7 EDL933 细菌将荧光素酶质粒放到 LB 琼脂板上, 其50µg/毫升卡那霉素从-80 摄氏度的库存。在37摄氏度培养 16-18 小时的细菌。</li>
	<li>选择一个单一的殖民地从隔夜板块和文化在3毫升 LB 培养基与50µg/毫升卡那霉素 16-18 小时在37摄氏度孵化器在 220 rpm。</li>
	<li>亚文化细菌 (1:100 稀释) 到卡那霉素 (50 µg/毫升) 含有 LB 汤 2.5-3 小时在37摄氏度孵化器 200 rpm。(例如, 添加2毫升的夜间培养细菌到200毫升 LB 培养基与卡那霉素 (50 µg/毫升) 补充)。</li>
	<li>孵育 2.5-3 小时的细菌, 并测量 600 nm (OD600) 的光学密度值, 直到该值介于0.9 到1之间。细菌细胞数的密度约为 108菌落形成单位 (CFU)/毫升。</li>
	<li>离心机重新培养的细菌在 8000 x g 30 分钟, 4 摄氏度。</li>
	<li>在不搅动细菌颗粒的情况下去除上清液, 用100毫升0.9% 无菌生理盐水用温和搅拌法清洗颗粒。</li>
	<li>重复步骤2.5 和2.6。</li>
	<li>清洗后, 离心机的细菌培养在 8000 x g 30 分钟, 4 摄氏度, 并轻轻地丢弃上清。</li>
	<li>将颗粒凝结成100倍, 0.9% 无菌生理盐水。例如, 如果200毫升细菌是离心, 添加2毫升正常生理盐水悬浮颗粒。</li>
	<li>确认细菌 CFU (它应该是大约 109 CFU/100 ul 在正常盐水冷凝后) 通过10倍的系列稀释25电镀浓缩细菌。</li>
</ol>3. 小鼠口服饲肠出血性大肠杆菌
<ol><li>用链霉素水 (5 克/升) 治疗6周大的雌性 C57BL/6 小鼠24小时。</li>
	<li>24小时后, 在饲前, 改用普通饮水24小时。用常规水治疗24小时后, 小鼠可以口服饲肠内大肠杆菌。</li>
	<li>通过拉回柱塞, 在注射器中填充 100 ul 肠出血性大肠杆菌。确保注射器内没有气泡。用手指对着注射器取出气泡。</li>
	<li>轻轻举起动物, 小心地放在笼子的顶端。</li>
	<li>小心地抓住老鼠的尾巴, 动物会抓住笼子的顶部, 试图离开。</li>
	<li>用拇指和食指抓住动物颈部和背部松弛的皮肤, 以防止老鼠头部移动, 轻轻地抑制老鼠。</li>
	<li>按住鼠标垂直位置插入饲针, 确保鼠标头固定和垂直。</li>
	<li>将饲针插入嘴后的小鼠嘴里, 向下移动到食道和胃部。</li>
	<li>当插入的针是在鼠标的一半或2/3 长度, 注射 100 ul 大肠杆菌, 其中包含约 109 CFU 细胞。</li>
</ol>4. 可视化
<ol><li>口服饲后, 检测1和2天的发光信号。</li>
	<li>在研究动物的生物发光信号之前, 麻醉将它们放入2.5% 异氟醚的腔内, 1.5 升/分钟氧。</li>
	<li>等待 2-5 分钟, 直到所有老鼠失去知觉, 停止移动。动物已经准备好体内检测生物发光。</li>
	<li>通过体内成像系统检测和成像动物的生物发光信号。请参阅5节 "数据采集" 软件操作。在成像过程中, 所有动物都在持续供应2.5% 异氟醚, 1.5 升/分氧。<ol>
<li>单击 "文件" > "实时", 然后手动将焦点放在鼠标上。</li>
		<li>选择曝光时间和激光强度。</li>
		<li>单击 "获取 > 捕获"。</li>
	</ol>
</li>
	<li>对于体外成像, 弄死小鼠颈椎脱位, 并清除感染小鼠的整个肠道. 用体内成像系统将肠道置于9厘米培养皿和图像中。该设置与 "体内" 映像相同, 但视图字段设置为-b。有关详细信息, 请参阅5节 "数据获取"。 注意: 颈椎脱位的方法: 用一只手抓住尾巴, 把笼子顶部的老鼠控制住, 这样动物就能抓住笼子。将标记笔或拇指和第一手指放在头骨底部的颈部后部。用手或物体快速向前推进, 同时抑制头部, 用手握住尾巴向后拉。 仔细检查以确认呼吸骤停, 没有心跳。</li>
</ol>5. 数据采集
注意: 用于数据采集的软件列在材料表中。
<ol><li>要获取图像, 请打开软件的购置控制面板 (图 2)。</li>
	<li>选择 "发光"、"照片" 和 "叠加"。</li>
	<li>将曝光时间设置为 "自动"。将 Binning 设置为 "介质"。</li>
	<li>设置ƒ/停止为1的发光和8的照片。ƒ/停止控制 CCD 探测器接收的光量。</li>
	<li>根据感兴趣的图像字段设置视图字段。选项 "D" 可以容纳五6周大的老鼠, 并在同一时间图像。"C" 可以在一个领域中想象三6周大的老鼠。</li>
	<li>一旦鼠标/样品准备好成像, 点击 "获取" 图像获取。</li>
	<li>打开已获取的图像数据。</li>
	<li>打开 "工具面板" 面板 (图 3)。</li>
	<li>选择 ROI 工具。我们建议圆圈 (最左边的一个) 来范围在图像上的发光区域 (图 4)。</li>
	<li>单击 "测量 ROIs" (铅笔图标) 以测量表面发光强度 (图 3)。"ROI 度量" 面板和量化值显示 (图 5)。</li>
	<li>使用 "ROI 度量" 面板左上角的 "配置测量" 来选择所需的值/信息 (图 6), 否则单击 "导出" 以导出此数据表并另存为. csv 文件 (图 5)。</li>
	<li>使用列 "总通量 (p/秒)" 的值作为. csv 文件中的发光强度量化。</li>
</ol>

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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-Tnl+0+transposon+derivatives+for+insertion+mutagenesis+and+gene+delivery+into+the+chromosome+of+Gram-negative+bacteria.">Mini-Tnl 0 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of Gram-negative bacteria.</a> Gene. 160 (1), 59-62 (1995).</li><li>Wiegand, I., Hilpert, K., Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agar+and+broth+dilution+methods+to+determine+the+minimal+inhibitory+concentration+(MIC)+of+antimicrobial+substances.">Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.</a> Nat Protoc. 3 (2), 163-175 (2008).</li><li>Pansare, V., Hejazi, S., Faenza, W., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+Long-Wavelength+Optical+and+NIR+Imaging+Materials:+Contrast+Agents,+Fluorophores+and+Multifunctional+Nano+Carriers.">Review of Long-Wavelength Optical and NIR Imaging Materials: Contrast Agents, Fluorophores and Multifunctional Nano Carriers.</a> Chem Mater. 24 (5), 812-827 (2012).</li><li>Heim, R., Cubitt, A. B., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+green+fluorescence.">Improved green fluorescence.</a> Nature. 373 (6516), 663-664 (1995).</li><li>Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clearer+vision+for+in+vivo+imaging.">A clearer vision for in vivo imaging.</a> Nat Biotechnol. 19 (4), 316-317 (2001).</li><li>Frangioni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+near-infrared+fluorescence+imaging.">In vivo near-infrared fluorescence imaging.</a> Current Opinion in Chemical Biology. 7 (5), 626-634 (2003).</li><li>Collins, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrobacter+rodentium:+infection,+inflammation+and+the+microbiota.">Citrobacter rodentium: infection, inflammation and the microbiota.</a> Nat Rev Microbiol. 12 (9), 612-623 (2014).</li><li>Mallick, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+murine+infection+model+for+Shiga+toxin-producing+Escherichia+coli.">A novel murine infection model for Shiga toxin-producing Escherichia coli.</a> J Clin Invest. 122 (11), 4012-4024 (2012).</li><li>Petty, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Citrobacter+rodentium+genome+sequence+reveals+convergent+evolution+with+human+pathogenic+Escherichia+coli.">The Citrobacter rodentium genome sequence reveals convergent evolution with human pathogenic Escherichia coli.</a> J Bacteriol. 192 (2), 525-538 (2010).</li><li>Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson , G. T., Farris, M. A., Roop, R. M., Peterson, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+new+derivatives+of+the+broad-host-range+cloning+vector+pBBR1MCS,+carrying+different+antibiotic-resistance+cassettes.">Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.</a> Gene. 166 (1), 175-176 (1995).</li><li>Galen, J. E., Nair, J., Wang , J. Y., Wasserman, S. S., Tanner, M. K., Sztein , M. B., Levine, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Plasmid+Maintenance+in+the+Attenuated+Live+Vector+Vaccine+Strain+Salmonella+typhiCVD+908-htrA.">Optimization of Plasmid Maintenance in the Attenuated Live Vector Vaccine Strain Salmonella typhiCVD 908-htrA.</a> Infect Immun. 67 (12), 6424-6433 (1999).</li><li>Francis, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+pneumococcal+infections+in+the+lungs+of+live+mice+using+bioluminescent+Streptococcus+pneumoniae+transformed+with+a+novel+gram-positive+lux+transposon.">Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon.</a> Infect Immun. 69 (5), 3350-3358 (2001).</li><li>Goldwater, P. N., Bettelheim, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+enterohemorrhagic+Escherichia+coli+(EHEC)+infection+and+hemolytic+uremic+syndrome+(HUS).">Treatment of enterohemorrhagic Escherichia coli (EHEC) infection and hemolytic uremic syndrome (HUS).</a> BMC Med. 10, (2012).</li></ol>

作者没有什么可透露的。

据报道, 用荧光素酶质粒转化出的肠出血性大肠杆菌已被用于检查其在宿主或基因表达式中的定位在体内8,11,12。在这里展示的小鼠模型也被报道来检测在小鼠宿主8中的肠大肠杆菌的殖民时间和定位。然而, 我们提供了如何管理肠内大肠杆菌疫苗接种小鼠灌的详细协议, 以及如何精心准备口腔饲的生物发?...

大肠杆菌 O157:H7 是一种病原体, 引起腹泻1, HS2, 溶血症3, 甚至急性肾功能衰竭4通过污染的水或食物。肠出血性大肠杆菌是一个致病性 enterobacterium 和殖民的胃肠道的人1。当出血性大肠杆菌第一次坚持宿主肠道上皮, 他们通过 III. 型分泌系统 (T3SS) 将定植因子注入宿主细胞, 该类型作为分子注射器诱导附加和谦逊 (a/E) 病变随后执行附着力 (殖民化)5。enterocyte 避嫌 (李) 致病性岛5的轨迹编码了这些涉及 A/E 病变形成的基因。
生物发光是一种光产生化学反应, 荧光素酶催化其基底荧光素生成可见光 6.这种酶法通常要求存在氧或三磷酸腺苷 (ATP)6。生物发光成像 (BLI) 允许研究人员的可视化和量化的寄主病原体相互作用的活动物7。BLI 可以通过以下生物发光细菌来表征活体动物的细菌感染周期, 它们迁移到并侵入不同的组织7;这揭示了感染的动态发展。此外, 动物的细菌负荷与生物发光信号8有关;因此, 简单直接地估计实验动物的病理状况是一种方便的指标。
这里使用的质粒包含荧光素酶操纵, luxCDABE, 这是从细菌Photorhabdus luminescens编码其自己的荧光素酶基板7,9。通过将这种荧光素酶表达质粒转化为细菌, 可以通过观察活动物中的这些生物发光细菌来监测其定植和感染过程。总体而言, BLI 和生物发光标记的细菌允许研究人员监测细菌数量和位置, 细菌的生存能力与抗生素/治疗治疗, 和细菌基因表达感染/殖民化6,7. 报告了许多致病细菌, 表达了luxCDABE操纵检查其感染周期和/或感染中的基因表达。这些细菌, 包括致肾盂肾炎大肠杆菌 10, 出血性大肠杆菌8, 11, 12, 13,致病大肠杆菌 (EPEC)8 , 柠檬酸杆菌rodentium14,15, 沙门氏菌伤寒16, 李斯特氏李斯特氏增生17, 耶尔森氏菌小肠结肠炎 18, 19,和霍乱弧菌20已被记录在案。
已开发了几个实验模型, 以促进研究肠大肠杆菌的殖民化在体外和体内21,22,23。然而, 缺乏适当的动物模型来研究肠内大肠杆菌的定植在体内, 从而导致缺乏细节。为促进对肠内大肠杆菌定植机制的研究,在体内, 建立动物模型来观察和量化活体动物的肠外培养是一种无创的方法是很有价值的。
这篇手稿描述了一个老鼠大肠杆菌的殖民模型, 使用一个生物发光表达系统来监测大肠杆菌在活寄主时间的殖民化。小鼠灌接种生物发光标记的大肠杆菌, 并在小鼠身上检测到无侵入性的体内成像系统13中的发光信号。感染了生物发光标记的大肠杆菌病的小鼠在2天后的肠道感染后显示出明显的生物发光信号, 这表明这些细菌在感染后2天后在宿主肠道内被殖民。前体图像数据表明, 这种殖民化在小鼠的盲肠和结肠中特别存在。利用该模型, 通过体内成像系统, 在活体宿主中检测出生物发光大肠杆菌的定植, 研究肠道细菌定植的详细机制, 从而促进对大肠杆菌诱发的生理和病理改变。

<table border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Shaker incubator</td>
			<td style="width:175px;">YIH DER</td>
			<td style="width:143px;">LM-570R</td>
			<td style="width:167px;">bacteria incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Orbital shaking incubator</td>
			<td style="width:175px;">FIRSTEK</td>
			<td style="width:143px;">S300</td>
			<td>bacteria incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">pBSL180</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>source of nptII gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pAKlux2</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>source of luxCDABE operon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T&#38;A Cloning Kit</td>
			<td>Yeastern Biotech</td>
			<td style="width:143px;">FYC001-20P</td>
			<td>use for TA cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nsi I</td>
			<td style="width:175px;">NEB</td>
			<td>R0127S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sca I</td>
			<td style="width:175px;">NEB</td>
			<td>R0122S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spe I-HF</td>
			<td style="width:175px;">NEB</td>
			<td>R0133S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sma I&#160;</td>
			<td style="width:175px;">NEB</td>
			<td>R0141S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">T4 ligase</td>
			<td style="width:175px;">NEB</td>
			<td style="width:143px;">M0202S</td>
			<td>use for plasmid cloning&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Ex Taq</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">10X Ex Taq Buffer</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP Mixture&#160;</td>
			<td style="width:175px;">TaKaRa</td>
			<td>RR001A</td>
			<td>use for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">PCR machine</td>
			<td style="width:175px;">applied Biosystem&#160;</td>
			<td style="width:143px;">2720 thermal cycler&#160;</td>
			<td>&#160;for PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Glycerol</td>
			<td style="width:175px;">SIGMA</td>
			<td style="width:143px;">G5516-1L</td>
			<td>use for bacteria stocking solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">NaCl</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">31434-5KG-R</td>
			<td>chemical for making LB medium, 10 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Tryptone</td>
			<td style="width:175px;">CONDA pronadisa</td>
			<td style="width:143px;">Cat 1612.00</td>
			<td>chemical for making LB medium, 10 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Yeast Extract powder</td>
			<td style="width:175px;">Affymetrix</td>
			<td style="width:143px;">23547-1 KG</td>
			<td>chemical for making LB medium, 5 g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Agar</td>
			<td style="width:175px;">CONDA pronadisa</td>
			<td style="width:143px;">Cat 1802.00</td>
			<td>chemical for making LB agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">kanamycin&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">K4000-5G</td>
			<td>antibiotics, use for seleciton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">streptomycin&#160;</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:143px;">S6501-100G</td>
			<td>antibiotics, eliminate the microbiota in mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">EDL933 competent cell</td>
			<td style="width:175px;">Homemade</td>
			<td style="width:143px;"></td>
			<td>method is on supplemental document&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Electroporator</td>
			<td style="width:175px;">MicroPulser</td>
			<td style="width:143px;"></td>
			<td>for electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Electroporation Cuvettes</td>
			<td style="width:175px;">Gene Pulser/MicroPulser</td>
			<td style="width:143px;">1652086</td>
			<td>for electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">High-speed centrifuge</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">Avanti, J-26S XP</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fixed-Angle Rotor</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">JA25.5</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Fixed-Angle Rotor</td>
			<td style="width:175px;">Beckman Coulter</td>
			<td style="width:143px;">JLA10.5</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge bottles</td>
			<td>Beckman Coulter</td>
			<td>REF357003</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge bottles</td>
			<td style="width:175px;">Thermo Fisher scientific</td>
			<td style="width:143px;">3141-0500</td>
			<td>use for centrifuging bacteria&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">eppendorf biophotometer plus&#160;</td>
			<td>eppendorf</td>
			<td>AG 22331 hamburg</td>
			<td>for measuring the OD600 value of bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">C57BL/6 mice&#160;</td>
			<td colspan="2">Laboratory Animal Center of NCKU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">lab coat, gloves</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>for personnel protection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane&#160;</td>
			<td>Panion &#38; BF Biotech Inc.</td>
			<td style="width:143px;">G-8669</td>
			<td>for mice anesthesia, pharmaceutical grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1ml syringe&#160;</td>
			<td style="width:175px;"></td>
			<td style="width:143px;"></td>
			<td>use for oral gavage of mice</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Reusable 22 G ball-tipped feeding needle</td>
			<td style="width:143px;">&#966;0.9 mm X L 50 mm</td>
			<td>use for oral gavage of mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">surgical&#160; scissors&#160;</td>
			<td></td>
			<td style="width:143px;"></td>
			<td>use for mice experiment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Xenogen IVIS 200 imaging system</td>
			<td>Perkin Elmer</td>
			<td>IVIS spectrum</td>
			<td>use for bioluminescent image capture&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Living Image Software</td>
			<td>Perkin Elmer</td>
			<td style="width:143px;">version 4.1</td>
			<td>use for quantifying the image data</td>
		</tr>
	</tbody>
</table>

detection of enterohemorrhagic escherichia coli colonization in murine host by non-invasive in vivo bioluminescence system

肠出血性大肠杆菌 (肠出血性大肠杆菌) O157:H7, 是 causesdiarrhea、出血性结肠炎 (HS) 和溶血尿毒症综合征 (溶血尿毒症) 的食源病原体, 可殖民到人类肠道。为了研究肠内大肠杆菌定植的详细机制在体内,必须有动物模型来监测和量化大肠杆菌的定植。通过将生物发光表达质粒转化为肠出血性大肠杆菌来监测和定量活寄主中大肠杆菌的定植, 我们在这里展示了一种小鼠大肠杆菌的定植模型。用生物发光标记的大肠杆菌接种的动物, 通过非侵入性的体内成像系统检测, 显示了小鼠强烈的生物发光信号。感染后1和2天后, 生物发光信号仍可在受感染的动物身上检测到, 这表明在寄主中至少有2天发生了大肠杆菌的殖民。我们还表明, 这些发光的肠外大肠杆菌定位到小鼠肠道, 特别是在盲肠和结肠, 从前体图像。这种鼠肠大肠杆菌定植模型可以作为一种工具, 以提高目前的知识, 肠出血性大肠杆菌的殖民机制。

我们通过口服饲对6周大的雌性 C57BL/6 小鼠进行生物发光标记的肠内毒素 (~ 109细菌细胞) 的管理。在1小时内对小鼠进行口服大肠杆菌疫苗接种后, 通过体内成像系统检查动物的荧光信号, 如图 7所示。结果表明, 饲小鼠具有生物发光标记的大肠杆菌的强烈发光信号。我们检查了2天后感染的信号。如图 8A所示, 接种了?...

Watch this Scientific Journal Video about Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System at JoVE.com

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

提出了一种利用生物发光标记细菌对肠出血性大肠杆菌 (大肠杆菌) 进行殖民化的小鼠模型的详细协议。在活体动物中, 通过非侵入性的体内成像系统来检测这些生物发光细菌, 可以提高我们目前对大肠杆菌定植的认识。

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 ...

detection-enterohemorrhagic-escherichia-coli-colonization-murine-host

Research

JoVE Journal

Immunology and Infection

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

9.4K 次观看.  National Cheng Kung University. 提出了一种利用生物发光标记细菌对肠出血性大肠杆菌 (大肠杆菌) 进行殖民化的小鼠模型的详细协议。在活体动物中, 通过非侵入性的体内成像系统来检测这些生物发光细菌, 可以提高我们目前对大肠杆菌定植的认识。

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

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

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. ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

无创<em&gt;体内</em炎性MMP活性的使用可激活荧光成像剂&gt;荧光光学成像

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

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. ...

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

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

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

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

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 ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

中性粒细胞中活性氧物种的实时定量 感染脑膜性 大肠杆菌

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...

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

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