使用生物发光成像对小鼠尿路感染及其治疗的纵向随访
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 96-well Black Flat Bottom Polystyrene Plate | Corning | 3925 | for in vitro imaging |
| Aesculap ISIS | Aesculap | GT421 | hair trimmer, with GT608 cap |
| Anesthesia vaporizer | Harvard apparatus limited | N/A | https://www.harvardapparatus.com/harvard-apparatus-anesthetic-vaporizers.html |
| Baytril 100 mg/mL | Bayer | N/A | Enrofloxacin |
| BD Insyte Autoguard 24 GA | BD | 382912 | Yellow angiocatheter, use sterile plastic tip for instillation |
| C57Bl/6J mice | Janvier | N/A | |
| Centrifuge 5804R | Eppendorf | EP022628146 | |
| Dropsense 16 | Unchained Labs | Trinean | to measure OD 600nm |
| Dulbecco's Phosphate Buffered Saline, Gibco | ThermoFisher Scientific | REF 14040-083 | |
| Ethanol 70% denaturated 5L | VWR international | 85825360 | |
| Falcon 14ml Round Bottom Polystyrene Tube, Snap-Cap | Corning | 352057 | |
| Falcon 50ml cellstart | Greiner | 227285 | |
| Hamilton GASTIGHT syringe, PTFE luer lock, 100 µL | Sigma-Aldrich | 26203 | to ensure slow bacterial instillation of 50 µL |
| Inoculation loop | Roth | 6174.1 | holder: Art. No. 6189.1 |
| Iso-Vet 1000mg/g | Dechra Veterinary products | N/A | Isoflurane |
| IVIS Spectrum In Vivo Imaging System | PerkinElmer | REF 124262 | imaging device |
| Kanamycine solution 50 mg/mL | Sigma-Aldrich | CAS 25389-94-0 | |
| Living Imaging Software | PerkinElmer | N/A | BLI acquisition software, version 4.7.3 |
| Luria Bertani Broth | Sigma-Aldrich | REF L3022 | alternatively can be made |
| Luria Bertani Broth with agar | Sigma-Aldrich | REF L2897 | alternatively can be made |
| Petri dish Sterilin 90mm | ThermoFisher Scientific | 101VR20 | to fill with LB agar supplemented with Km |
| Pyrex Culture flask 250 mL | Sigma-Aldrich | SLW1141/08-20EA | |
| Slide 200 Trinean | Unchained Labs | 701-2007 | to measure OD 600nm |
| UTI89-lux | N/A | N/A | Generous gift from Prof. Seed |
| Vortex | VWR international | 444-1372 |
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