在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌
Summary
肺炎链球菌是导致病原体引起严重的社区获得性肺炎，负责超过200万世界各地的死亡。的牵连健身或致病细菌因素的影响，可以在实时中采用发光细菌急性小鼠肺炎或菌血症模型来监测。
Abstract
肺炎是发展中国家的主要医疗保健问题和工业化国家之一，具有相当的发病率和死亡率。尽管在知识这个疾病，重症监护病房（ICU）的可用性，并使用有效的抗菌药物和有效的疫苗的进步，死亡率仍然很高1。 肺炎链球菌是社区获得性肺炎的病原体领先（CAP）和菌血症的人类最常见的原因之一。这种病菌配备的表面暴露的黏附和毒力因素导致肺炎和侵入性肺炎球菌疾病（IPD）的医疗设备。细菌健身或毒力因子在体内的作用进行评估是最重要的解开S。肺炎链球菌致病机制。肺炎，菌血症和脑膜炎的小鼠模型已被用于确定肺炎球菌因素在昼夜温差的影响ferent感染的阶段。在这里，我们描述了一个协议来监控实时传播肺炎球菌在小鼠鼻腔或腹腔内感染发光细菌之后。该结果表明，繁殖和传播的肺炎球菌的下呼吸道和血液，从而可以可视化并进行评价使用成像系统和所附的分析软件。
Introduction
由病毒或细菌呼吸道感染仍然是全球最常见的社区获得性或临床问题引起全球所有死亡的大约三分之一之一。关键细菌物种是流感嗜血杆菌和肺炎链球菌 2。然而，这些细菌种类是通常的自然呼吸道菌群的常见成分。因此，细菌马车是一定风险的侵入性疾病，并根据免疫状况或个人的倾向也。无症状定植被触发侵袭性感染。 肺炎链球菌是社区获得性肺炎（CAP）和菌血症人类最常见的原因之一，主要病原体。在健康个体S。肺炎链球菌 （肺炎球菌）往往是上呼吸道，在那里他们都面临着非致病性细菌无症状和无害殖民者常住菌也与病原体如嗜血杆菌属的。或金黄色葡萄球菌和人的免疫防御系统的第一道防线。携带率最高的是年幼的孩子（37％），甚至在拥挤的日间护理中心（58％）高3-5。最年轻的人群和老人，通过空气传播的载体和鼻咽部分泌物6接受肺炎球菌，属于使用的肺炎球菌结合疫苗（PCV10或PCV13在儿童和23价多糖PPSV23成人）一个高危人群和疫苗接种建议在美国（美国）和许多欧洲国家4。在PPSV23涵盖负责〜90％的菌血症性肺炎球菌疾病在美国和欧洲，预防成人从而有效地侵入性肺炎球菌疾病（IPD）的血清型，而PCVS涵盖儿童中最常见的血清型。因此，瞳距由于疫苗的种类（VT）是热镀CED但非疫苗血清型显示高毒力潜力及耐药性已经出现4,7-12。鼻咽部的水库是起点肺炎蔓延到鼻窦或中耳引发有害的局部感染。更重要的是，肺炎球菌直接通过气道蔓延至支气管和肺部造成威胁生命的第4,13。肺部感染常常伴随着组织和屏障的破坏，从而使病原体传播到血液中，并造成IPD。 CAP和IPD的发病率最高，免疫功能低下者或在4,13年代的极端。负责从共生与高致病病原体转换的情况仍在争论。然而，除了改变宿主的易感性和进化适应伴随着较高的毒力和增加的抗生素抗性已建议对PNE至关重要的影响umococcal感染14-16。
病原体被赋予了黏附素介导的亲密接触粘膜上皮细胞的多样性。克服气道黏液后，肺炎球菌粘附于宿主细胞是通过表面暴露粘附素与细胞受体相互作用的直接并利用细胞外基质成分或血清蛋白的桥联分子4,17,18便利。多才多艺的病原体肺炎球菌还配备了参与宿主免疫防御机制回避的因素。此外，它们具有适应各种宿主milieus如肺，血液和脑脊液（CSF），分别为5,17,19,20的能力。
细菌因素对发病机制和炎性宿主反应的影响进行了研究中的肺炎，菌血症实验动物模型中，或脑膜炎21-25。尽管是人类的病原体，这些模型是我们LL成立的破译肺炎球菌组织嗜性，致病机制，或肺炎球菌疫苗候选保护性。近交系小鼠品系的遗传背景决定了易患肺炎。鼻内感染了肺炎球菌免疫BALB / c小鼠，结果为：耐腐蚀，而CBA / Ca和SJL小鼠抗肺炎球菌感染22更容易。这意味着，类似于人类的遗传背景和宿主防御机制确定感染的结果。因此，有必要进一步努力解开抗性基因座的小鼠不容易受到肺炎球菌感染的基因组中。该发现导致了变化的体内毒性的协议。而不是过去常常使用的近交系BALB / c小鼠后，极易受到CD-1/MF1远交小鼠品系人现在通常用来研究的功能丧失的肺炎球菌的致病性或适用性的因素26-28的效果。此外，可用生物发光性肺炎和光学成像技术的允许感染的实时生物发光生物成像。在肺炎球菌的优化luxABCDE基因盒（质粒保罗的TN 4001 luxABCDE公里R）已经插入到染色体中通过转座子诱变的单一整合位点。生物发光肺炎链球菌已被用来评估短少在致病性或适用性的因素及其易位从一个解剖部位到另一个26,28-31肺炎球菌突变体的衰减。
在这里，我们提供了一个协议，用于在小鼠肺炎或败血症模型肺炎球菌感染的生物成像。放大和传播生物发光肺炎球菌在鼻内或腹膜内感染的小鼠可以很容易地随着时间的推移，使用光学成像系统和相同的动物在不同时间点监测。
Protocol
Representative Results
Discussion
在动物身上进行的所有实验必须由地方当局和伦理委员会的批准。在体内实验感染的细菌负荷在感染动物的各种宿主龛，必须在不同时间点在感染后确定。在这些实验条件的动物具有从血液，鼻咽，bronchoalvelar灌洗或器官如肺，脾和脑细菌的隔离之前被牺牲掉。计算每个主机小生细菌的数量，并评估细菌因素对毒力的影响时，血液或器官必须由细菌分离随之恢复和电镀在固体培养基上?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
研究在实验室被授予由德意志研究联合会（DFG医管局3125/3-2，DFG医管局3125/4-2）和德国联邦教育与研究部（BMBF）医疗感染基因组学（FKZ 0315828A）到上海的支持。
Materials
| Todd Hewitt broth | Carl Roth, Karlsruhe, Germany | X936.1 | |
| Yeast extract | Carl Roth, Karlsruhe, Germany | 2363.2 | |
| Blood agar plates | Oxoid, Wesel, Germany | PB5039A | |
| Kanamycin | Carl Roth, Karlsruhe, Germany | T832.2 | |
| Erythromycin | Sigma-Aldrich,Taufkirchen, Germany | E6376 | |
| fetal bovine serum (FBS) | PAA Laboratories, Coelbe, Germany | A11-151 | |
| CD-1 mice, female | Charles River, Sulzfeld, Germany | CD1SIFE06W08W | female CD-1 mice, six to eight weeks old |
| Ketamin 500mg, Curamed injection solution | Schwabe-Curamed, Karlsruhe, Germany | ||
| Rompun 2%, injection solution | Bayer Animal Health, Monheim, Germany | ||
| BD Plastipak 1 ml syringes | Becton Dickinson, Heidelberg, Germany | 300015 | sterile Luer-Lok™ syringes with needle |
| Gel Loader Tips | peqlab | 81-13790 | MµltiFlex™ Tips |
| Hyaluronidase | Sigma-Aldrich | H3884-100mg | Hyaluronidase Type IV-S from Bovine test |
| Oxygen | Air Liquide, Düsseldorf, Germany | M1001L50R2A001 | |
| Isofluoran | Baxter, Unterschleißheim, Germany | ||
| pGEM-T Easy | Promega, Mannheim, Germany | ||
| Oligonucleotides | Eurofins MWG, Ebersberg, Germany | ||
| Qiaprep Spin Midiprep Kit | Qiagen, Hilden, Germany | 27104 | |
| PCR DNA purification kit | Qiagen, Hilden, Germany | 28106 | |
| Equipment | |||
| Living Image 4.1 software | Caliper Life Sciences/PerkinElmer, Rodgau, Germany | ||
| XGI-8 Gas Anesthesia System | Caliper Life Sciences/PerkinElmer, Rodgau, Germany | ||
| IVIS Spectrum Imaging System | Caliper Life Sciences/PerkinElmer, Rodgau, Germany | ||
| Biophotometer | Eppendorf AG, Hamburg, Germany |
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