Name: longitudinal follow-up of urinary tract infections and their treatment in mice using bioluminescence imaging
Uploaded: 2021-06-14T13:30:00
Description: Watch this Scientific Journal Video about Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging at JoVE.com

这项工作得到了法兰德斯研究基金会（FWO Vlaanderen;G0A6113N），鲁汶大学研究委员会（C1-TRPLe;T.V.和W.E.）和 VIB（到电视）。W.E.是法兰德斯研究基金会（FWO Vlaanderen）的高级临床研究员。菌株UTI89-lux是Seed教授实验室13的慷慨礼物。

所有动物实验均按照欧盟共同体理事会指南进行，并得到KU Leuven动物伦理委员会的批准（P158 / 2018）。
1. 培养细菌（适应于7、13、14）
<ol><li>制备<ol>
<li>选择最适合实验需求的发光UPEC菌株。 注意：在这里，选择临床膀胱炎分离物UTI89（大肠杆菌），因为它在人类和啮齿动物中的泌尿致病能力，以...

<ol>
	<li>Foxman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+urinary+tract+infections:+incidence,+morbidity,+and+economic+costs.">Epidemiology of urinary tract infections: incidence, morbidity, and economic costs.</a> American Journal of Medicine. 113 (1), 5-13 (2002).</li><li>O'Brien, V. P., Hannan, T. J., Nielsen, H. V., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+and+vaccine+development+for+the+treatment+and+prevention+of+urinary+tract+infections.">Drug and vaccine development for the treatment and prevention of urinary tract infections.</a> Microbiology Spectrum. 4 (1), 1128 (2016).</li><li>Nielubowicz, G. R., Mobley, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+in+urinary+tract+infection.">Host-pathogen interactions in urinary tract infection.</a> Nature Reviews Urology. 7 (8), 430-441 (2010).</li><li>Foxman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+urinary+tract+infection.">The epidemiology of urinary tract infection.</a> Nature Reviews Urology. 7 (12), 653-660 (2010).</li><li>Carey, A. J., 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=Urinary+tract+infection+of+mice+to+model+human+disease:+Practicalities,+implications+and+limitations.">Urinary tract infection of mice to model human disease: Practicalities, implications and limitations.</a> Crititical Reviews in Microbiology. 42 (5), 780-799 (2016).</li><li>Barber, A. E., Norton, J. P., Wiles, T. J., Mulvey, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+model+systems+for+the+study+of+urinary+tract+infections+and+related+pathologies.">Strengths and limitations of model systems for the study of urinary tract infections and related pathologies.</a> Microbiology and Molecular Biology Reviews. 80 (2), 351-367 (2016).</li><li>Hung, C. S., Dodson, K. W., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+urinary+tract+infection.">A murine model of urinary tract infection.</a> Nature Protocols. 4 (8), 1230-1243 (2009).</li><li>Contag, C. H., 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=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Molecular Microbiology. 18 (4), 593-603 (1995).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nature Medicine. 4 (2), 245-247 (1998).</li><li>Doyle, T. C., Burns, S. M., Contag, C. H. <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+bioluminescence+imaging+for+integrated+studies+of+infection.">In vivo bioluminescence imaging for integrated studies of infection.</a> Cellular Microbiology. 6 (4), 303-317 (2004).</li><li>Hutchens, M., Luker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+bioluminescence+imaging+to+the+study+of+infectious+diseases.">Applications of bioluminescence imaging to the study of infectious diseases.</a> Cellular Microbiology. 9 (10), 2315-2322 (2007).</li><li>Avci, 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=In-vivo+monitoring+of+infectious+diseases+in+living+animals+using+bioluminescence+imaging.">In-vivo monitoring of infectious diseases in living animals using bioluminescence imaging.</a> Virulence. 9 (1), 28-63 (2018).</li><li>Balsara, Z. R., 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=Enhanced+susceptibility+to+urinary+tract+infection+in+the+spinal+cord-injured+host+with+neurogenic+bladder.">Enhanced susceptibility to urinary tract infection in the spinal cord-injured host with neurogenic bladder.</a> Infection and Immunity. 81 (8), 3018-3026 (2013).</li><li>Huang, Y. Y., 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=Antimicrobial+photodynamic+therapy+mediated+by+methylene+blue+and+potassium+iodide+to+treat+urinary+tract+infection+in+a+female+rat+model.">Antimicrobial photodynamic therapy mediated by methylene blue and potassium iodide to treat urinary tract infection in a female rat model.</a> Scientific Reports. 8 (1), 7257 (2018).</li><li>Mulvey, M. A., Schilling, J. D., Hultgren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+persistent+Escherichia+coli+reservoir+during+the+acute+phase+of+a+bladder+infection.">Establishment of a persistent Escherichia coli reservoir during the acute phase of a bladder infection.</a> Infection and Immunity. 69 (7), 4572-4579 (2001).</li><li>Hannan, T. J., Hunstad, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+for+E.+coli+urinary+tract+infection.">A murine model for E. coli urinary tract infection.</a> Methods in Molecular Biology. 1333, 83-100 (2016).</li><li>Hopkins, W. J., Gendron-Fitzpatrick, A., Balish, E., Uehling, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+and+host+responses+to+Escherichia+coli+urinary+tract+infection+in+genetically+distinct+mouse+strains.">Time course and host responses to Escherichia coli urinary tract infection in genetically distinct mouse strains.</a> American Society for Microbiology. 66 (6), 2798 (1998).</li><li>Zhang, Y., 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=Efficacy+of+Nonsteroidal+Anti-inflammatory+Drugs+for+Treatment+of+Uncomplicated+Lower+Urinary+Tract+Infections+in+Women:+A+Meta-analysis.">Efficacy of Nonsteroidal Anti-inflammatory Drugs for Treatment of Uncomplicated Lower Urinary Tract Infections in Women: A Meta-analysis.</a> Infectious Microbes &amp; Diseases. 2 (2), 77-82 (2020).</li><li>Vanherp, L., 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=Sensitive+bioluminescence+imaging+of+fungal+dissemination+to+the+brain+in+mouse+models+of+cryptococcosis.">Sensitive bioluminescence imaging of fungal dissemination to the brain in mouse models of cryptococcosis.</a> Disease Models &amp; Mechanisms. 12 (6), 039123 (2019).</li><li>Keyaerts, M., Caveliers, V., Lahoutte, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging:+looking+beyond+the+light.">Bioluminescence imaging: looking beyond the light.</a> Trends in Molecular Medicine. 18 (3), 164-172 (2012).</li><li>Marques, C. N., Salisbury, V. C., Greenman, J., Bowker, K. E., Nelson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrepancy+between+viable+counts+and+light+output+as+viability+measurements,+following+ciprofloxacin+challenge+of+self-bioluminescent+Pseudomonas+aeruginosa+biofilms.">Discrepancy between viable counts and light output as viability measurements, following ciprofloxacin challenge of self-bioluminescent Pseudomonas aeruginosa biofilms.</a> Journal of Antimicrobial Chemotherapy. 56 (4), 665-671 (2005).</li><li>Vande Velde, G., Kucharikova, S., Van Dijck, P., Himmelreich, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+increases+in+vivo+screening+efficiency+for+antifungal+activity+against+device-associated+Candida+albicans+biofilms.">Bioluminescence imaging increases in vivo screening efficiency for antifungal activity against device-associated Candida albicans biofilms.</a> International Journal of Antimicrobial Agents. 52 (1), 42-51 (2018).</li><li>Oliver, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+findings+on+the+viable+but+nonculturable+state+in+pathogenic+bacteria.">Recent findings on the viable but nonculturable state in pathogenic bacteria.</a> FEMS Microbiology Reviews. 34 (4), 415-425 (2010).</li><li>Kucharikova, S., Van de Velde, G., Himmelreich, U., Van Dijck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+biofilm+development+on+medically-relevant+foreign+bodies+in+a+mouse+subcutaneous+model+followed+by+bioluminescence+imaging.">Candida albicans biofilm development on medically-relevant foreign bodies in a mouse subcutaneous model followed by bioluminescence imaging.</a> Journal of Visualized Experiments: JoVE. (95), e52239 (2015).</li><li>Van de Velde, G., Kucharikova, S., Schrevens, S., Himmelreich, U., Van Dijck, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+non-invasive+monitoring+of+pathogen-host+interactions+during+Candida+albicans+biofilm+formation+using+in+vivo+bioluminescence.">Towards non-invasive monitoring of pathogen-host interactions during Candida albicans biofilm formation using in vivo bioluminescence.</a> Cellular Microbiology. 16 (1), 115-130 (2014).</li></ol>

与 CFU 计数相比，BLI 的优势 纵向数据 传统上计算CFU以量化微生物负荷的方法的一个主要缺点是需要 死后 器官均质，每只动物只能提供一个横截面数据点。相反，BLI能够对受感染的动物进行非侵入性纵向随访。这些动物每天可以成像2到3次，从而提供对感染动力学的详细见解。此外，对同一动物的重复测量大大减少了充分动力实验所需的动物数量。此外，研究人员可以专?...

尿路感染（UTI）是人类最常见的细菌感染之一。几乎一半的女性在其一生中会出现有症状的UTI1。局限于膀胱的感染可引起泌尿系统症状，例如尿频增加、尿急、血尿、尿失禁和疼痛。当感染上升到上尿路时，患者会出现肾盂肾炎，伴有不适、发热、寒战和背痛。此外，高达20%的UTI患者患有复发性感染，导致抗生素敏感性急剧下降2，3，4。近年来，人们对治疗和预防复发性UTI的新疗法越来越感兴趣。尽管对下尿路的先天性和适应性免疫以及侵袭和定植所需的细菌毒力因素有了更好的了解，但治疗方案的根本性变化尚未转化为日常泌尿外科实践2。为了研究UTI的发病机制和体内的易感性，一种可重复的、非侵入性的测定是必不可少的。
已经描述了从线虫到灵长类动物的多种动物UTI模型，但小鼠模型主要使用5，6。该模型包括（雌性）小鼠的经尿道导管插入术，随后将细菌悬浮液（最常见的是泌尿致病性大肠杆菌（UPEC））直接滴注到膀胱腔7中。接种后，细菌负荷传统上通过确定菌落形成单位（CFU）来量化。该技术需要牺牲动物以获得死后器官均质和连续稀释，从而限制了数据输出和可重复性。此外，使用这种技术不可能对个体动物的细菌负荷进行纵向随访。
1995年，Contag等人建议使用生物发光标记的病原体来监测活体动物的疾病过程8，9。从那时起，生物发光成像（BLI）已应用于许多感染模型，如脑膜炎，心内膜炎，骨髓炎，皮肤和软组织感染等10，11，12。在小鼠UTI模型中，可以使用来自Photorhabdus发光的具有完全勒克斯操纵子（luxCDABE）的UPEC菌株13。酶促反应由细菌荧光素酶催化，其依赖于长链醛的氧化，在氧气存在下与还原的黄素单核苷酸反应，产生氧化的黄素，长链脂肪酸和光12。lux操纵子编码荧光素酶和合成底物所需的其他酶。因此，所有代谢活性细菌将连续发射蓝绿色（490nm）光，而无需注射外源性底物12。lux标记细菌发射的光子可以使用高灵敏度的冷却电荷耦合器件（CCD）相机捕获。
在UTI模型中使用生物发光细菌允许对细菌负荷进行纵向，非侵入性定量，而不需要在CFU测定的随访期间在固定时间点牺牲动物。尽管存在广泛的可能性，积累了这种BLI技术在其他领域的稳健性及其相对于UTI经典模型的优势的证据，但它尚未在UTI研究中得到广泛实施。这里介绍的方案提供了详细的分步指南，并强调了BLI在未来所有UTI研究中的优势。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well Black Flat Bottom Polystyrene Plate</td>
			<td>Corning</td>
			<td>3925</td>
			<td>for in vitro imaging</td>
		</tr>
		<tr>
			<td>Aesculap ISIS</td>
			<td>Aesculap</td>
			<td>GT421</td>
			<td>hair trimmer, with GT608 cap</td>
		</tr>
		<tr>
			<td>Anesthesia vaporizer</td>
			<td>Harvard apparatus limited</td>
			<td>N/A</td>
			<td>https://www.harvardapparatus.com/harvard-apparatus-anesthetic-vaporizers.html</td>
		</tr>
		<tr>
			<td>Baytril 100 mg/mL</td>
			<td>Bayer</td>
			<td>N/A</td>
			<td>Enrofloxacin</td>
		</tr>
		<tr>
			<td>BD Insyte Autoguard 24 GA</td>
			<td>BD</td>
			<td>382912</td>
			<td>Yellow angiocatheter, use sterile plastic tip for instillation</td>
		</tr>
		<tr>
			<td>C57Bl/6J mice</td>
			<td>Janvier</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5804R</td>
			<td>Eppendorf</td>
			<td>EP022628146</td>
			<td></td>
		</tr>
		<tr>
			<td>Dropsense 16</td>
			<td>Unchained Labs</td>
			<td>Trinean</td>
			<td>to measure OD 600nm</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline, Gibco</td>
			<td>ThermoFisher Scientific</td>
			<td>REF 14040-083</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70% denaturated 5L</td>
			<td>VWR international</td>
			<td>85825360</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 14ml Round Bottom Polystyrene Tube, Snap-Cap</td>
			<td>Corning</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50ml cellstart</td>
			<td>Greiner</td>
			<td>227285</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton GASTIGHT syringe, PTFE luer lock, 100 &#181;L</td>
			<td>Sigma-Aldrich</td>
			<td>26203</td>
			<td>to ensure slow bacterial instillation of 50 &#181;L</td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Roth</td>
			<td>6174.1</td>
			<td>holder: Art. No. 6189.1</td>
		</tr>
		<tr>
			<td>Iso-Vet 1000mg/g</td>
			<td>Dechra Veterinary products</td>
			<td>N/A</td>
			<td>Isoflurane</td>
		</tr>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>REF 124262</td>
			<td>imaging device</td>
		</tr>
		<tr>
			<td>Kanamycine solution 50 mg/mL</td>
			<td>Sigma-Aldrich</td>
			<td>CAS 25389-94-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Living Imaging Software</td>
			<td>PerkinElmer</td>
			<td>N/A</td>
			<td>BLI acquisition software, version 4.7.3</td>
		</tr>
		<tr>
			<td>Luria Bertani Broth</td>
			<td>Sigma-Aldrich</td>
			<td>REF L3022</td>
			<td>alternatively can be made</td>
		</tr>
		<tr>
			<td>Luria Bertani Broth with agar</td>
			<td>Sigma-Aldrich</td>
			<td>REF L2897</td>
			<td>alternatively can be made</td>
		</tr>
		<tr>
			<td>Petri dish Sterilin 90mm</td>
			<td>ThermoFisher Scientific</td>
			<td>101VR20</td>
			<td>to fill with LB agar supplemented with Km</td>
		</tr>
		<tr>
			<td>Pyrex Culture flask 250 mL</td>
			<td>Sigma-Aldrich</td>
			<td>SLW1141/08-20EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide 200 Trinean</td>
			<td>Unchained Labs</td>
			<td>701-2007</td>
			<td>to measure OD 600nm</td>
		</tr>
		<tr>
			<td>UTI89-lux</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Generous gift from Prof. Seed</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR international</td>
			<td>444-1372</td>
			<td></td>
		</tr>
	</tbody>
</table>

longitudinal follow-up of urinary tract infections and their treatment in mice using bioluminescence imaging

尿路感染 （UTI） 是人类最常见的细菌感染之一，常规使用经验性抗生素治疗。然而，由于微生物耐药性的增加，最常用的抗生素的疗效已经下降。为了找到替代治疗方案，非常需要更好地了解UTI的发病机制和确定UTI易感性的机制。为了在动物模型中研究这一点，研究UTI病程的可重复，非侵入性测定是必不可少的。
多年来，细菌负荷计数的金标准一直是确定特定样品体积的菌落形成单位（CFU）。该技术需要 死后 器官均质和连续稀释，限制了数据输出和可重复性。作为替代方案，生物发光成像（BLI）越来越受欢迎，以确定细菌负荷。使用 lux 操纵子标记病原体，可以以非侵入性方式进行灵敏的检测和定量，从而实现纵向随访。到目前为止，BLI在UTI研究中的采用仍然有限。
本手稿描述了BLI在小鼠尿路感染模型中的实际实施。这里提供了培养细菌，膀胱内滴注和成像的分步指南。检查了 体内 与CFU的相关性，并通过比较未经处理的受感染动物与抗生素处理动物的细菌负荷来提供概念验证。此外，还讨论了 在体内 UTI模型中实施BLI的优点，局限性和注意事项。BLI在UTI研究领域的实施将极大地促进UTI发病机制的研究以及预防和治疗UTI的新方法的发现。

体内 BLI与滴注时接种物的CFU相关。 为了评估BLI在体内的检测限以及与接种物CFU的相关性，以不同浓度的UTI89-lux和PBS作为阴性对照感染小鼠。在滴注之前，扫描未受感染的动物以确定背景发光。随后的图像在滴注后立即获得（图1A）。在滴注UTI89-lux后，生物发光在2 x 104 CFU以上被强烈地检测到，并且建立了接种物?...

Watch this Scientific Journal Video about Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging at JoVE.com

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

本手稿描述了使用 勒克斯 操纵子膀胱内施用泌尿道致病细菌以诱导小鼠尿路感染，以及随后使用生物发光成像对细菌负荷进行 纵向体内 分析。

使用生物发光成像对小鼠尿路感染及其治疗的纵向随访

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, ...

菌落形成单位的枚举限制了UTI领域研究的可重复性。生物发光成像将推进膀胱生理学、UTI发病机制和易感性的体内UTI研究。生物发光成像能够进行纵向随访，实时了解感染的演变。此外，它大大减少了所需的动物数量。生物发光成像有助于研究复发性或慢性感染，这在人类中很常见。此外，研究人员可以使用生物发光成像识别上升的感染或传播到血液中。通过在补充了硫酸卡那霉素的LB板上用接种环将甘油菌储备条纹出，并在37摄氏度下培养过夜来获得单个菌落。填充无菌的14毫升聚苯乙烯圆底管，用双位置卡扣帽和五毫升LB汤补充硫酸卡那霉素。选择具有接种环的单个细菌菌落，并将其添加到LB肉汤中。涡旋10秒钟，以确保正确混合。静培与卡扣帽在37摄氏度的开放位置过夜。孵育后，涡旋管10秒，以确保细菌培养物的适当均质化。通过向25毫升不含抗生素的新鲜LB培养基中加入25微升细菌悬浮液，在Erlenmeyer中进行亚培养。关闭 Erlenmeyer 和培养物静态过夜。在安装当天，将Erlennmeyer的培养物倒入50毫升培养管中，然后离心。倾析上清液并将细菌沉淀重悬于10毫升无菌PBS中。再次涡旋管10秒钟。选择接种物的实验浓度，并使用标准曲线确定600纳米处的相应OD。将一毫升重悬的细菌培养物加入9毫升无菌PBS中，并调整直至达到所需的OD 600纳米。将无菌的24号血管导管尖端安装在100微升注射器上。用准备好的细菌溶液填充注射器。将一只动物放在仰卧位的工作表面上，并在安装过程中使用鼻锥保持稳定的异氟醚麻醉。涂抹眼药膏。通过轻柔按压并在耻骨上区域进行打圈来排出残留的尿液，然后用70%乙醇清洁下腹部。用生理盐水润滑导管尖端。将食指放在腹部的非惯用手上，然后轻轻向上推。以90度角垂直开始尿道导管插入术。遇到阻力后，在进一步插入之前将其水平倾斜。缓慢安装50微升的细菌接种物。安装完成后，将注射器和导管保持在适当的位置几秒钟，然后缓慢缩回以防止泄漏。每个实验组使用一根导管。将动物置于鼻锥的仰卧位置，以准备成像，并使用每个实验组的一根导管准备成像。如有必要，施用抗生素或实验性药物，如文本手稿中所述。打开BLI采集软件，单击成像设备中的初始化，以测试相机和载物台控制器系统，并将电荷耦合设备相机冷却至零下90摄氏度。单击采集，自动保存到，选择发光和照片。通过将激发滤镜设置为"阻止"并将发射滤光片设置为"打开"来检查默认发光设置。拍摄第一张图像时，将曝光时间设置为自动。对于体内测量和明亮信号，请将曝光时间设置为30秒左右。如果由于图像饱和而出现警告，请缩短曝光时间。选择中等分档，F/停止，然后选择正确的FOV。对小鼠进行成像时，将受试者高度设置为一厘米。同时成像长达五英里，并使用光挡板将动物分开以防止反射。关闭门并单击"获取"以开始成像序列，然后填写有关实验的详细信息。将小鼠从成像室中取出并将它们放回笼子中。麻醉后检查是否完全恢复，然后将笼子放回通风架，直到下一个成像周期。启动映像软件，然后单击"浏览"加载实验文件。使用工具调色板调整图像的色阶。使用 ROI 工具在图像上绘制感兴趣的区域，确保该区域足够大以覆盖整个区域，并对所有图像使用相同的尺寸。然后单击ROI测量以量化光强度。安装后立即获得的小鼠的后续图像显示，在20， 000个菌落形成单位以上可以强烈地检测到，并且建立了接种物的菌落形成单位与体内生物发光之间的线性相关性。在没有接受任何治疗的对照组中研究了尿路感染的自然演变。在一组抗生素治疗的动物中详细可视化了抗生素治疗对感染动力学的影响。在第一剂恩诺沙星之后，通过总光子通量测量，细菌负荷立即降低。没有一只经过处理的动物随后的细菌负荷增加。使用对数转换总光子通量曲线下的面积计算每种动物的总细菌负荷，显示用恩诺沙星处理的动物和未处理的动物在10天的时间过程中存在显着差异。这些结果提供了概念证明，BLI可用于评估UTI感染动力学的差异。生物发光成像是一种非侵入性技术，可以与其他方法结合使用，例如定期收集尿液样本进行细菌或生化分析或实验。在这里，我们已经证明，生物发光成像是评估尿路感染病程的新治疗策略的强大工具。

Research

JoVE Journal

Immunology and Infection

Transurethral Induction of Mouse Urinary Tract Infection

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

Using Luciferase to Image Bacterial Infections in Mice

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

生物发光成像的NADPH氧化酶活性在不同的动物模型

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

生物发光成像检测到非洲锥虫病的晚期感染

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

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

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, ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

在抗菌蓝光治疗的体内研究的多药耐药鲍曼不动杆菌感染烧伤利用生物发光成像

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

基于显微 CT 和荧光分子层析成像的多模态成像方法对博莱霉素诱导的小鼠肺纤维化的纵向评价

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

尿道感染小鼠模型产生的单细胞内细菌群落的分离, 用于下游单细胞分析

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...