JH要感谢，感谢导师，朋友和前经理瓦莱丽·贝瑞的第一个教我们这种模式;和加里Woodnutt，谁教我们的基础，我们的灵感抗菌PK / PD领域的承诺。所有的作者要感谢大卫·佩恩对他的支持和赞赏我们的科学。我们要感谢我们谁使用这些方法，尤其是皮特DeMarsh，罗布·斯特劳布，罗尼页面和尼莉莎西蒙生成数据的身体贡献体内微生物的同事前者。最后，我们要感谢我们的体外微生物同事们的援助，特别是提供我们所要求的MIC（麦克洛斯基琳，乔什西和莎朗敏）;并为我们的临床微生物的团队谁提供专家建议和支持（琳达米勒，妮可Scangarella，阿曼和德博拉·巴特勒）。

S. pneumonie 所有的程序都是按照由葛兰素史克机构动物护理和使用委员会（IACUC）批准的方案，并达到或超过美国实验室动物护理的认证（AAALAC），卫生和人类的美系的标准服务和所有地方和联邦动物福利法律。 注意:除非另有规定，应使用无菌技术来执行的所有过程。 1.培养细菌分离<ol><li>制备3至6琼脂肺炎链球菌或流感嗜血板，如肉汤培养一般不接种提供足够的材料。 <ol><li>在-80℃下移除存储冷冻的储存培养，完全解冻，和条纹高达每板100微升到补充有羊血（ 肺炎链球菌 ）的胰蛋白酶大豆琼脂或到巧克力琼脂（ 流感嗜血菌 ），使用标准的微生物学技术。在37℃下具有或不具有5％CO 2孵育过夜。 </li></ol></li><li>准备肉汤培养与肺炎克雷伯菌 ， 铜绿假单胞菌和鲍曼不动杆菌。 <ol><li>在-80℃拆下存储冷冻储存培养，完全解冻，和分装100微升的股票为50毫升脑心脏浸液（BHI）肉汤。在37℃轻微振荡（大约120rpm下）孵育过夜。 </li><li> [可选]如果日志阶段文化需要，分装1毫升从所得的过夜培养成50毫升新鲜BHI肉汤。在37℃轻微振荡（大约120rpm下）孵育3小时。 </li></ol></li></ol> 2.准备盐水菌悬液<ol><li>所有悬浮液的准备步骤（2.1到2.3），利用无菌盐水在环境空气和37℃之间的温度。刮落肺炎链球菌或流感嗜血杆菌的隔夜嘉实增长从琼脂平板从用无菌环的表面上。收获的材料转移到5毫升无菌生理盐水，直到多云，得到不透明的悬挂，轻轻涡直至均匀。 </li><li>离心机50毫升的肺炎克雷伯菌 ， 铜绿假单胞菌和鲍曼不动杆菌用于接种最终肉汤培养物（ 如，隔夜或登录阶段培养）为以4,500×g的5分钟。用吸液管和丢弃上清。重悬在至少5毫升的无菌盐水的沉淀，并轻轻涡旋以获得均匀的悬浮液。 </li><li>如果需要的话，进一步稀释该悬浮液用无菌盐水，以获得在8至9日志10菌落形成单位（CFU）/ mL的范围内的密度。测定CFU /毫升删除等分。连续稀释并如第7.5和7.6中所述镀等分试样。 </li></ol> 3.准备琼脂接种体<ol><li>准备营养琼脂雅一小瓶（约50毫升）定制造商的说明书或熔化的瓶子被预先制备并存储固体营养琼脂的。让其冷却至约50℃。 </li><li>输送液体琼脂和所有必要的工具和设备的感染过程到动物会在被感染的程序区。保持一小的水浴中的区域设定在42℃。 </li><li>允许琼脂以达到约50℃的温度，然后等分9毫升到无菌管适当尺寸以适合水浴中。离开该管在水浴直到琼脂平衡到约42℃。确保水是在深度将覆盖在其容器中的琼脂的表面，但不接触帽或盖。 </li><li>来自步骤2.3中添加1毫升的盐水细菌悬浮液入含有在水浴9毫升琼脂试管。帽筒，颠倒几次混合，并返回到水洗澡。 </li></ol> 4.nesthetize，插管，接种动物注意:无特定病原体（SPF）免疫雄性SD大鼠，体重100 - 120 g或雄性CD-1小鼠体重20 - 24 g的推荐。提供食物和水随意所有动物上和社会上容纳它们木屑或与标准的12小时光照-黑暗周期其它吸收床上用品。 <ol><li>以进行任何实验之前，获取并消毒适当大小的金属套管和聚乙烯管材的动物物种。获得无菌1毫升一次性注射器和一次性无菌25号针头。 注意:始终处置这些注射器和针头的利器容器。 <ol><li>对于大鼠，购买或制造的金属套管是约12厘米的长度，为0.9的ID - 1.0毫米，1.0外径 - 1.2毫米，并弯成一个近似45°角在一端。切断的聚乙烯管的11至12英寸的长度为0.4毫米，外径0.8mm的标识。 （参见<stroNG&gt;图1A。 ） </li><li>对于老鼠，购买20＃动物喂食管。通过用钳子把持它和急剧拉除去从管的端部的球，然后将端轻轻弯曲以近似45°角。切断的聚乙烯管的11至12英寸长度为0.28毫米和外径0.61毫米的ID。还购买并消毒的玻璃100μL的微注射器和30号金属微注射针。 （参考图1B）。 </li><li>将金属套管在玻璃，螺丝帽管和高压用标准方法进行消毒。浸泡和存储削减聚乙烯管材的长度在有盖的烧杯中含有酒精。 </li></ol></li><li>准备工作表面和气管插管设备。 <ol><li>将一个洁净的一次性垫到工作面上，靠近水浴。转移金属套管，在其玻璃储存管，该表面。取下存储管帽和滑动cannu的"把手"末端拉至管的开口端。 </li><li>使用无菌镊子，从烧杯中取出聚乙烯管中的一个的长度，并通过无菌金属套管的内侧插入，并确保其自由移动。通过滑动针数毫米入软管适合的无菌的一次性25号针头（老鼠）或玻璃微注射器的无菌的30号金属针（小鼠）到聚乙烯管的自由端。 （参见图1A为大鼠和图1B为只小鼠）。 </li><li>同时在金属套管和聚乙烯管处导引标记通过以下在单个实施安乐死的动物后述的插管程序，以指示插入在动物的深度。打开胸腔进行观察，然后将金属套管完全和适当超前的聚乙烯管（如第4.7节）。使用了不可磨灭的笔，标出有它满足了动物和聚口的金属套管乙烯管，其中它满足了金属套管的顶端，以帮助在其余的动物妥善安置。 </li></ol></li><li>内通风橱（或使用适当的清除剂体系），直到咽反射被抑制（约1分钟）麻醉多达6只动物在同一时间在1.5升/分钟的氧气与≤5％异氟烷供给的密闭室。 注意:在此之前进行任何实验，建立了安全剂量和暴露时间为异氟烷正在使用的特定条件下（ 例如，大小/类型腔与动物的尺寸/物种/株），以防止无意的致死照射。 </li><li>通过将无菌尖端到盐水和向后拉柱塞填充的无菌一次性1毫升注射器（老鼠）用无菌盐水。填充无菌玻璃微注射器（小鼠）如下所述。附加注射器装配到聚乙烯管针，并通过管道，直到syring冲洗盐水的总体积e的清空。 <ol><li>填补微注射器（小鼠），完全除去从注射器的金属柱塞和针（装配到聚乙烯管），并同时设置预留。 </li><li>填充无菌的一次性1ml注射器用无菌生理盐水（如上所述），并附加一个一次性无菌25号针头。针插入微注射器（其中金属柱塞将被插入）的顶部，并按下一次性注射器的柱塞直到填满微注射器用生理盐水。 </li><li>丢弃被用于填充微注射器成尖锐件容器中的一次性注射器和针头。金属微注射针（装配到聚乙烯管）重新连接到微注射器的前端和重新插入金属柱塞压下直至盐水的总体积已通过注射器和管冲洗。 </li></ol></li><li>装满基础琼脂接种注射器来回通过只是直到管被引（ 例如完全充满琼脂）的管中的M水浴的容器，冲洗琼脂。 <ol><li>对于大鼠，请从一次性注射器25号针头（安装到聚乙烯管材）。丢弃在一个锐器容器所使用的注射器。通过将无菌尖端到接种物和向后拉柱塞填充的新的无菌的一次性1毫升注射器从水浴容器中的基于琼脂接种物。通过按压只是直到管被完全充满琼脂柱塞重新连接针（装配到聚乙烯管）和冲洗琼脂过管路。 </li><li>对于小鼠，取出金属微注射针（装配到聚乙烯管）和金属柱塞从微注射器，待用。使用无菌的一次性1ml注射器和无菌的一次性25号针，从在容器中填充微注射器与基于琼脂接种使用第4.4节中描述的填充过程水浴。重新装上金属微注射针（装配到聚乙烯管），插入金属柱塞和冲洗琼脂通过管仅仅直到管被完全充满琼脂。 </li></ol></li><li>从麻醉室中取出一只动物。放置动物在与头部向右和尾部向左一次性垫仰卧位置， 如图1C所示 。 </li><li>使用插管装置（ 即配有聚乙烯管的金属套管），插管动物和插入插管插入大叶左肺（ 见图1D）。 <ol><li>插入金属套管的自由端插入动物的嘴。转动套管，以使自由端被向上成角度，并将其轻轻地推进进气管，小心地绕过喉结构有轻微的扭转运动。确认插入气管相对于通过在触诊与左食指的气管环， 如图1C轻轻滑动套管稍微向前和向后几次食道。 </li><li>当所述套管到达那里的气管分成左和右支气管分岔，使朝向动物的左侧轻微扭转运动，以确保插管被插入左支气管。推进金属套管直到结束是中途到四分之三向下左肺，使用先前放置向导标志以确认适当的深度已经达到。 </li><li>有了金属套管，推进聚乙烯管材几个毫米。使用的管道的先前放置向导标志，以确保它前进仅足够远以退出金属套管的端部和未刺破肺。 </li></ol></li><li>有了这两个套管，使用附带的注射器灌输琼脂suspe 100微升（老鼠）或20μL（小鼠）nsion深入到大叶左肺（ 见图1D）。撤销聚乙烯管材的几个毫米，然后轻轻取出完整的气管插管设备。将它放回玻璃储存管，动物移动到一个新的笼子里恢复。 </li><li>继续麻醉，插管和接种剩余的动物。 <ol><li>麻醉动物的批次间，除去从注射器针头（装配到管件）。对于大鼠，丢弃在尖锐件容器中的使用过的一次性注射器，并从在水浴接种物填充一个新的无菌的一次性注射器。对于小鼠，笔芯从使用第4.4节中描述的填充技术水浴接种相同的玻璃微注射器。 </li><li>如果琼脂开始在气管插管设备勾芡，通过冲洗管道的所有剩余琼脂。从注射器（离开装配到聚乙烯管针）取出针。按照介绍的步骤4.4节通过插管设备冲洗温暖，无菌生理盐水，重复必要完全畅通无阻。如第4.5节所述，从注射器清空所有盐水和再次准备琼脂接种。 </li><li>计算每个使用从盐水悬浮液中确定的CFU动物的最终细菌接种物（见2.3节），盐水悬浮液进入琼脂（ 如十倍）的最终稀释倍数，以及灌输到动物（ 例如 ，100微升的体积老鼠或20微升小鼠）。 </li></ol></li></ol> 5.评估动物势误接种或其他不良事件<ol><li>插管时仔细观察所有动物，琼脂灌输和恢复从麻醉中评估潜在的错误接种。 （根据当地IACUC准则），其出血任何动物立即安乐死，表现出异常呼吸（例如呼吸困难或喘气），不动一般约为笼，或不会出现明亮的眼睛和健康的在从麻醉中苏醒。 </li><li>如果发生呼吸的自发停止（不放在通常当接种足够深并阻塞气道），通过观察确认死亡并执行安乐死的辅助方法（如颈椎脱位或开胸）。 </li><li>最小化时间的动物的长度暴露在异氟醚，如麻醉只需要几分钟，以执行该过程。如果使用注射麻醉剂，确保动物在观察，直到完全清醒。使用注射麻醉剂时放置动物，尤其是小鼠，在温热环境进行恢复。 </li><li>明显的呼吸，毛发竖立，减少的活性和降低体温:在研究期间对于呼吸系统疾病下列体征每天至少两次观察动物。立即安乐死（根据当地IACUC准则）任何动物是奄奄一息，经验用NCES呼吸困难或呼吸窘迫，有一个明显的体温降低后的处理，是不能或不愿动，或者不能达到食物和水。 </li></ol> 6.辖治疗试验<ol><li>准备并根据规划研究设计管理试验治疗。开始治疗给药间隔1〜2小时后感染（或更高的基线细菌负荷需要进一步延误治疗）。无法给出准备和试验治疗管理的具体细节，因为它是取决于研究的目的以及每个个体化治疗的属性。 （请参考图3，例如，） </li><li>留出一组感染，未经处理的动物作为基线控制。列举在这些动物在当时处理的肺中的细菌负荷的其它组被启动时，按照第7概述的程序。 </li><li>对于PK / PD分析，评估在血液和/或感染的动物的组织中施用处理（多个）的药代动力学曲线。对药代动力学研究的具体过程是本文的范围之外。 </li></ol> 7.从肺部枚举细菌存活<ol><li>安乐死一组未经处理的动物中的处理是在研究期间（通常为24，48或96小时后感染）通过吸入暴露结束启动的其他组（基线）和所有剩余的动物的时间逐渐上升碳的水平所推荐的本地IACUC准则的密闭室或另一种方法中氧化钛。经观察确认死亡，并执行安乐死的辅助方法（如颈椎脱位或开胸）。 </li><li>放在一个干净的一次性垫子仰卧位的动物，彻底润湿了皮毛用酒精胸前。 </li><li>用无菌剪刀划破皮肤，下面的肌肉层和肋骨平行于胸骨打开胸腔。轻轻抓住用无菌镊子肺部并拉出，用剪刀在必要时将它们从气管中分离出来。将肺到无菌纱布吸去多余的血液。如果心脏也被删除，剖析它拿走并丢弃。 </li><li>使用无菌剪刀以暴露内部段，并使用无菌镊子放置肺（加上被无意中剪掉任何片）放入实验室混合器袋的底部肺小剪刀。简单地说滚厚的圆形物体（如钢笔或标记）在包包混搭组织的外部。无菌生理盐水入袋的吸移管1毫升，将包成一个实验室混合器，并运行在高速实验室搅拌器2分钟。 </li><li>传送从混合袋匀浆样品成用吸管管或板。通过aliquotting 1部分匀浆成9份盐水稀释匀浆十倍（如100微升匀浆入900微升萨兰E）。稀释由所得悬浮液以类似的方式再次十倍。继续被另一个十倍因子稀释每种新生成的悬浮液，直到至少6连续稀释液已经从原始匀浆制备。 </li><li>每个从所有稀释到补充有羊血胰胨豆胨琼脂平板上20微升（ 肺炎链球菌，肺炎克雷伯，铜绿假单胞菌或鲍曼不动杆菌 ），或到巧克力琼脂平板（ 流感嗜血菌 ）吸移管式三份等分试样。在37℃有或无的 CO 2孵育过夜。 （参见图1E为所得板的一个例子。） </li><li>算在每个三次重复的菌落在包含"可数"数（ 即没有太多菌落合理计数）的第一次稀释。计算三次重复的平均值。由50（平均值乘以占电镀总1毫升20微升确定每个肺CFU匀浆）和保在从该菌落计数原始匀浆最终稀释因子。 </li></ol>

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作者是公司内的所有支付股票葛兰素史克制药公司的员工和自己的股份。

对于复制本感染模型最优的成功，以下补充建议应予以考虑。在一项研究中在体内的利用之前的3倍-新菌株毒力可以传代2得到增强。冷冻细菌股票应始终从主体内衍生来源与编制从原来的几段越好，重新冻结或解冻的股票重用气馁。使用最近从患者恢复肺炎临床分离，和/或准备在数生长期的文​​化也可以帮助改善建立感染。一个五倍稀释到琼脂（ 例如 2毫升盐水悬浮液加入到8mL的琼脂）可以用来代替的十倍，以增加细菌接种物，并接种物体积可以调节基于动物的大小（ 例如 200微升/动物通常接种到250克只）。之前加入盐水细菌悬浮液进琼脂（ 即在步骤2.3），细菌密度可以预测或通过视觉检查，麦克法兰标准或光密度测量估计。是有帮助的熟悉的体外生长特性的每个之前的体内实验进行隔离。一致性在生长和处理能为任何给定的分离的细菌密度的最准确的估计。从所描述的那些，如替代媒体和组织均化不同标准的微生物学方法，可以适当使用用于制备接种物，并从感染组织列举细菌。 强烈建议准备或实验前融化的琼脂白天和设定为50℃，一个单独的水浴中过夜存放。这将简化流程，并帮助防范琼脂处于感染时太热。 41温度 - 42℃，达到maintai之间的良好平衡宁琼脂在液体状态而不为短期的细菌生存太热。作为一种可能的替代营养琼脂，贵金属琼脂已成功在一些实验中使用。琼脂接种物的一管通常是足够用于整个实验（和建议），但多个管可用于感染大量的动物来制备（ 例如 ，大于60），或者当感染过程需要超过30更长- 45 min如果多个接种管是必需的，盐水细菌悬浮液添加到琼脂每个管，因为它是需要的。这减少了细菌存活和/或健身将由长期暴露的影响，以在接种前的高温下的风险。应当注意的是，使用多种菌管还可能需要额外的动物的随机化过程。只要有可能，从相同的接种物制剂感染所有的动物。建议的实验的大小调整到熟练的当前级别与德chnique。 一个熟练的科学家可以插管，在不到30秒的接种动物和接种长达5 -每批次6只动物（ 即一个注射器充满琼脂接种）。对于那些学习技术，速度是琼脂重要开始固化;然而，接种的精确放置是更重要的。每批次1或2只动物开始，并且随着技术变得更加熟悉。动物继续插管呼吸;因而，对于接种的时间窗口取决于动物从异氟烷，这应该是大约2如何迅速恢复 - 3分钟。过程中所唤起的动物可以重新麻醉并试图在第二时间，但它不建议如此反复做。在第一次尝试成功的接种预期在动物中的几乎100％的一次熟练度实现。注意，它是比较容易首先用大鼠的学习技术;小鼠更细腻，也更小LS更容易与琼脂凝固堵塞如果不迅速操纵。预变暖注射器，管道和盐水以及保持在一个温暖的（不热）表面上的插管装置，例如在低设置一个加热垫，可以帮助防止琼脂凝固。当学习的技术中，它也有利于用深色染料（如浓缩亚甲蓝）代替琼脂的细菌悬浮液来实施接种的正确放置。执行该过程的描述，但安乐死动物立即染料接种以下（不使动物麻醉并实施安乐死之间恢复）。解剖，以确定其中的染料已经被放置，并相应地调整技术。 此模型中的主要终点是CFU从感染肺部。生存不是细菌负担一个很好的指标，而不是推荐端点。对于功效研究，所建议的N是5 - 每组6只动物，因为这是predicted可检测≥1日志组之间10 CFU差异至少有90％的电力。动物可在基团被感染，使得笼配合保持在一起，或真正的随机化过程可以遵循。需要注意的是，如果基团被笼分配，基团应当以随机顺序感染感染最后结束的研究生长对照（确认细菌存活/健身并不受的时间暴露于高温下的长度在水浴）。没有数据截尾的方法应该是必要的，并且没有推荐使用立即除去该已明显误接种在研究开始时（之前的任何治疗开始）的任何动物的除外。被之前的研究结束时处死的动物应该采样并包括在数据集除非排除有效的原因是前瞻性鉴定结果（ 即无关的感染或治疗事件）。药物残留可能AFF等一些样品，并在所有的体内感染模型的一个重要的考虑因素。如果一个化合物给出频繁，靠近安乐死的时间，或具有长的半衰期，也可以是存在于所述组织匀浆以足够高的浓度，以继续在细菌枚举过程杀死细菌体外 （稀释和电镀过夜温育过程中在琼脂平板的样品或）。为了防止这种情况，活性炭和/或其降解的活性分子，而不伤害细菌细胞可以添加到之前均化样品中的添加剂。其它方法包括离心样品以除去大部分的活性化合物（应留在上清液）的和使用不同的稀释和电镀方案充分稀释的活性化合物到非抑制浓度。 如由图2中所示的例子中，该模型成功我nduces在啮齿类动物肺部感染具有广泛的生物体包括那些不能很好地在其它模式（ 例如流感嗜血杆菌 ）生长的。这些感染是一致的和可重复的，从而减少了实验需要，由于进行多次，以模型故障和/或性能差与给定的分离的可能性。虽然动物是免疫活性，它们无法迅速解决感染，如果在所有。这允许在研究长度增加的灵活性，因为许多分离株通过至少96小时保持的可行感染，而不需要重复注射以维持中性粒细胞减少。研究在免疫动物的抗菌功效的潜在益处已如前所述20，21，并且有证据表明，对某些化合物（ 如恶唑烷酮），从非中性白细胞减少症的啮齿动物的数据可以更精确地与这些呈现中性粒细胞减少相比预测人类接触的目标5。 T的多功能工具他模型是表现在图3和4及表1和2这些研究已与该模型生成的，以支持引线优化努力16,17的大集合公布和未公布的数据，22-24的一部分，用于比较并提出了人类给药确认服法19，25-29和PK / PD表征18，30-32。而这种模式是最初更复杂的鼻内吸入法相比进行，有如上所述的许多好处。随着实践和日常使用，这些技术应该成为易于执行。 它可能在一些研究中，在基线细菌负荷比通常针对其他肺部感染模型下加以注意。这是由于在很大程度上所需稀释成琼脂和小挑战体积，尤其是在小鼠。然而，还应当注意该细菌生长被认为在即使当初始负担是相当低的所有情况。目标基线细菌负荷是6至6.5日志10 CFU /肺（6.8至7.3日志10单位/克，基于平均肺重量的小鼠组织的），它对应于在人类患者中，估计重症肺炎33密度。较高的基线负担可以通过进一步浓缩细菌接种物或者通过延缓化合物给药开始，以允许附加的细菌生长来实现;然而，越来越多的挑战负荷过多，可导致严重的异常和急性疾病（ 即死亡不到24小时），这是难治所有抗菌治疗，无论敏感性。虽然接种最初放置优先到动物的左肺，感染通常传遍两肺。进肺部疾病，细菌对其他器官的传播，并最终发病率往往是OBSERVED与肺炎链球菌，肺炎克雷伯菌和铜绿假单胞菌 。有趣的是，与流感嗜血杆菌和鲍曼不动杆菌感染通常更包含的，很少导致死亡在规定的细菌接种。 从这个模型得到疗效结果与体外药敏简介以及定义PK / PD的目标密切相关。在细菌负荷减少例行所考虑易受剂被测试菌株观察到的，而那些被认为是耐展览高于基线16，17，19，24-29无变化或细菌生长。已在大鼠评价两个喹诺酮类和使用此模型32大环内酯类研究显示与基线与和，更重要的是在中性粒细胞减少小鼠中确定的目标相关联相比，对于所要求的PK / PD目标1登录肺炎链球菌 10减少，具有社区获得性细菌pneum临床指标onia 6，7，34，35。 Gepotidacin，一种新的机制抗生素，是对多个肺炎链球菌测试并需要一个类似的PK / PD目标对该肺部感染模型31 1-日志10减少作为在中性粒细胞减少大腿模型1日志10还原所需36时肺部穿透被考虑（对文件数据）。同样，在小鼠对绿脓杆菌 GSK2251052确定的PK / PD目标是为瘀，1年和2记录此肺模型30和中性粒细胞减少大腿感染模型之间的CFU降低10时一致穿透肺部被认为是37，38 。使用鼻内接种是穷人一个中性粒细胞减少肺部感染模型的相关性;然而，GSK2251052没有产生比在这项研究中38静态响应多。作为与6日志相比，这可能是归因于较高的细菌接种物，这是8日志10 CFU /小鼠<sub&gt; 10 CFU /鼠标在中性粒细胞减少大腿37和气管插管30款。如这个数据的收集为基准关键的，因为它允许用现有的模型和临床数据相关的直接比较，以评估所述平移预测能力。更广泛地使用气管内插管肺部感染模型将这些类型的分析提供额外的数据。 有此免疫肺炎模型的一些限制。首先，它不能很好地适合于由于高细菌接种物通常需要在太严重感染这样的研究结果评价自发耐药性的出现。以下企图达到7或8的日志10 CFU在基线，快速发病率已经观察到的细菌负担（ 即动物，在不到24小时变得垂死）尽管接种的彻底清洗以除去（在文件中的数据）预先存在的毒素。它可能通过使用较大的啮齿动物来克服这个问题。鼠，尤其较重大鼠（&gt; 250克），似乎更好地耐受更高的接种小鼠相比，并且可以是适合于这些类型的研究。第二个限制是，使用琼脂作为感染增强剂可以排除使用用于某些宿主的评价模型:病原体相互作用。据认为，琼脂提供受保护的焦点，直到感染肺部内完全建立。有可能提供在盐水中，而不是琼脂，以帮助克服这个问题的接种物然而，应当注意的是，这只会产生感染与某些菌株。第三，要成为精通技术需要一个陡峭的学习曲线。该过程可以是微妙的，特别是在小鼠。这是很容易的金属套管误放入食道，并且过大的力可能导致要么食道或气管穿刺。接种仔细放置也要求或动物可能根本无法恢复或得不到充分的感染。然而，有耐心和毅力，可以成为高度熟练和快速执行的技术，顺利和准确。 通过去除中性粒细胞减少的要求，增加了鲁棒性和再现性，允许调查研究更病原体和分离，提高实验设计的灵活性，并提供一个具有挑战性的感染来表征药效学肺炎，这种免疫活性的肺部感染模型增加重大价值的抗生素发现社区。增加使用这种模式通过额外的调查将有助于提供必要的基准为它赢得更广泛的接受和继续提供适当的解释的支持信息。

在评估感染的动物模型中潜在的候选药物的疗效抗菌药物发现过程的关键组成部分。 体内功效研究用于提供重要的数据的决策横跨发现努力的跨度，从阐明结构-活性关系（SAR），并通过确定的药物代谢动力学，药效学（PK / PD）优化引线化学系列化合物的特性和支承剂量选择为临床开发和监管部门批准。众多动物感染模型在文献中对这些目的描述。其中最常见和广泛使用的模型是中性粒细胞减少小鼠大腿感染，这是由鹰1,2率先后来由Vogelman和克雷格3,4扩大。这种模式是非常宝贵的支持细菌药敏断点的决心和提供PK / PD指标来指导人类的剂量。有许多考试普莱斯这个模型的预测能力已经被证明4-7。然而，大腿感染模型的缺点之一是缺乏直接对肺部感染的相关性。现在有一种更加强调感染的动物模型的感染人类的​​站点的站点匹配;因此，为了研究肺炎的治疗化合物，理想的是利用动物肺部感染模型。 已经描述和用于抗菌功效研究包括鼻内吸入，通过口咽路线抽吸，气雾剂曝光和外科气管内接种8用于诱导在实验室动物的肺部感染的方法。在许多情况下，将动物（特别是小鼠）必须首先，以实现一个坚固的肺部感染呈现中性粒细胞减少。尽管这样严重免疫状态，从而产生可行的感染在啮齿类动物的细菌病原体和应变数量有限。例如，它可以是难以成功建立流感嗜血杆菌或鲍曼不动杆菌在肺炎模型9，10，和甚至更致命的病原体，如肺炎链球菌，铜绿假单胞菌和肺炎克雷伯氏菌，可以带来挑战11-13。 1991年，史密斯在其感染是由通过一个简单的非手术气管插管法直接14灌输细菌进入肺部免疫建立大鼠断乳描述的肺部感染模型。浆果等 。后来修改了感染小鼠的15方法。使用基于琼脂接种物，这种接种技术诱导既免疫大鼠和小鼠肺炎链球菌，流感嗜血，肺炎克雷伯，绿脓杆菌和鲍曼不动杆菌健壮肺部感染。它是适合于广泛的菌株，包括那些窝藏各种抗蚀剂ANCE因素和那些不通过其他途径接种产生一个可行的感染。它也允许在免疫动物来确定功效，条件比用于患者群体是不严重免疫传统中性粒细胞减少小鼠模型更相关。在多个病原体和分离该模型的一致性和可靠性使得它非常适合抗菌功效的研究。

<table><tbody><tr><td>TSA II Trypticase Soy Agar with 5% Sheep Blood</td><td>Becton Dickinson and Company</td><td>4321261</td><td></td></tr><tr><td>Chocolate II Agar</td><td>Becton Dickinson and Company</td><td>4321267</td><td></td></tr><tr><td>BBL Brain Heart Infusion Broth Modified</td><td>Becton Dickinson and Company</td><td>299070</td><td></td></tr><tr><td>BBL Trypticase Soy Broth with 20% Glycerol</td><td>Becton Dickinson and Company</td><td>297808</td><td>storage media for frozen stock</td></tr><tr><td>Inoculating loop</td><td>Nunc</td><td>251586</td><td></td></tr><tr><td>0.9% Sodium Chloride, USP</td><td>Baxter Healthcare Corporation</td><td>NDC 0338-0048-04</td><td></td></tr><tr><td>Difco Nutrient Agar</td><td>Becton Dickinson and Company</td><td>213000</td><td></td></tr><tr><td>30 mL free standing centrifuge tube with cap</td><td>EverGreen Scientific</td><td>222-3530-G80</td><td></td></tr><tr><td>50 mL Polypropylene Conical Tube 30 x 115 mm</td><td>Falcon, a Corning Brand</td><td>352070</td><td></td></tr><tr><td>14 mL Polypropylene Round-Bottom Tube 17 x 100 mm</td><td>Falcon, a Corning Brand</td><td>352059</td><td></td></tr><tr><td>Guide Tool</td><td>Intek Services Ltd</td><td>GT01</td><td>custom-made metal cannula for rats</td></tr><tr><td>90&#039; Portex tubing</td><td>SAI</td><td>POR-080-100</td><td>plastic cannula for rats</td></tr><tr><td>1 mL TB syringe slip tip</td><td>Becton Dickinson and Company</td><td>309659</td><td></td></tr><tr><td>PrecisionGlide Needle 25 G x 5/8</td><td>Becton Dickinson and Company</td><td>305122</td><td></td></tr><tr><td>Animal feeding needle - straight 20 x 3&quot; 2-1/4</td><td>Popper and Sons, Inc</td><td>7903</td><td>used to create metal cannula for mice</td></tr><tr><td>Intramedic polyethylene tubing</td><td>Clay Adams brand - Becton Dickinson and Company</td><td>427401</td><td>plastic cannula for mouse&amp;nbsp;</td></tr><tr><td>75 TN 5.0 &amp;micro;L syringe 26s/2&quot;/2</td><td>Hamilton</td><td>87930</td><td>HPLC glass injection syringe</td></tr><tr><td>Pyrex tube, culture 25x150 screwcap with rubber liner</td><td>Corning</td><td>9825-25</td><td>to autoclave/store metal cannulae</td></tr><tr><td>70% isopropyl alcohol</td><td>Vi-Jon (Swan)</td><td>NDC 0869-0810-43</td><td></td></tr><tr><td>Isoflurane, USP</td><td>Piramal Healthcare</td><td>NDC 66794-013-10</td><td></td></tr><tr><td>Stomacher&amp;nbsp;</td><td>Seward</td><td>Stomacher80</td><td></td></tr><tr><td>Stomacher 80 classic bags</td><td>Seward</td><td>BA6040</td><td></td></tr><tr><td>Assay plate, 96 well, round bottom</td><td>Costar, Corning Inc</td><td>3795</td><td>optional, for serial diluting</td></tr><tr><td>Blood Omni Plates</td><td>Hardy</td><td>#A127</td><td>optional, rectangular blood plates</td></tr></tbody></table>

a robust pneumonia model in immunocompetent rodents to evaluate antibacterial efficacy against s. pneumoniae, h. influenzae, k. pneumoniae, p. aeruginosa or a. baumannii

候选抗菌治疗功效必须在感染的动物模型中表现为发现和开发过程的一部分，优选地在其模拟预期的临床适应症的模型。描述了一种用于在免疫大鼠和小鼠诱导健壮的肺部感染的方法，它允许对治疗引起的肺炎链球菌 ， 流感嗜血杆菌 ， 绿脓杆菌 ， 肺炎克雷伯或鲍曼不动杆菌严重肺炎的模型的评估。动物麻醉，并基于琼脂接种物深存入通过非手术气管插管肺。所得感染是一致的，可重复的，并且为至少48小时的稳定和高达96小时对大多数的菌株。已经与市售抗菌剂的研究表明良好的相关体内效力和体外敏感性，并测定药物动力学/药效目标之间的一致性在这个模型中和临床认可目标已经观察到。虽然有学习技术时的初始时间的投入，它可以快速和有效地一旦熟练实现执行。该模型的优点包括的中性粒细胞减少需求消除，提高了稳定性和可重复性，学习更多的病原体和隔离，在研究设计和建立一个具有挑战性的感染更高的灵活性在免疫活性宿主的能力。

所需要的工具，该动物的取向，和插管的深度在图1中示出。还示出菌落上连续稀释和肺匀浆样品的一式三份的电镀后的琼脂板上生长的代表性实例。在上述基线控制多个日志10 CFU的细菌负担的增加对于大多数细菌菌株感染肺部通常观察到的，即使在96小时后感染，与动物之间的变异为低（ 图2）。对于某些流感嗜血 ，可能有小的增长;然而，感染不应开始自我解决一个48或96小时的时间段（ 图2B）内。这种肺部感染模型可以铅化学系列优化过程中被用来支持的SAR， 如图3的两个不同的系列16，17代表的化合物。它提供了一个一致的，可重复的满足HOD用于评估免疫动物的PK / PD和用于确定在与新的多肽脱（PDF）的功效相关的最小抑制浓度（fAUC / MIC）的浓度-时间曲线下游离药物领域抑制剂对抗肺炎链球菌和流感嗜血杆菌 （ 图4）。此外，fAUC / MIC为瘀和目标1登录CFU减少10从横渡11 肺炎链球菌和5 流感嗜血杆菌株（ 表1）18确定基线控制。耦合用药物递送系统本感染模型来重新人类药代动力学曲线的大鼠创建用于向临床研究前评估人类给药方案的有力工具。这方面的例子总结于表2，这表明阿莫西林-克拉维酸的缓释制剂比现有剂量方案与的Eleva生物体更有效泰德MIC值19。 <img alt="图1" src="/files/ftp_upload/55068/55068fig1.jpg" /> 图1: 非手术气管插管。工具示于插管的大鼠（A）和小鼠（B）。一旦麻醉，如图℃，用头部向右和尾部向左如果执行的技术中的个人是右旋的动物应定位。轻轻地放在喉咙左食指，以帮助触诊气管环正确插管位置的确认。接种物应放置深入到左肺，如图大鼠肺（D）的腹面观。菌落的样品的连续稀释和电镀后在琼脂生长应该出现类似于E中所示。图1D，版权所有©吉尔·史密斯[实验动物，第25卷（1991年），G·史密斯，A49] 14 -支气管内滴入建立大鼠呼吸道感染，46页简单的非手术方法。转载许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55068/55068fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55068/55068fig2.jpg" /> 图2: 在感染的动物的肺细菌生长的代表性实例。平均值±选自N = 5只动物/组的标准偏差CFU数据示出了用于肺炎链球菌 （A）， 流感嗜血杆菌 （B）中 ， 铜绿假单胞菌 （C）中 ， 肺炎克雷伯 （D）和鲍曼不动杆菌的各种菌株（E）。每对（浅灰色）的第一棒是基线bacte在1或2小时后感染里亚尔负担，而第二杆是在48小时后感染（深灰色）或96小时后感染（黑色）的结束研究生长控制。没有数据截尾的方法被应用;因此，这些结果包括来自每个组的所有5只动物的数据。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55068/55068fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55068/55068fig3.jpg" /> 图3: 铅优化过程中评估各种化工系列的功效。使用所描述的技术肺部感染模型用来探索结构 - 活性关系作为非细菌性IIA型拓扑异构酶方案的一部分。有前途的化合物7和1票反对评价喹诺酮敏感（A）16或喹诺酮耐药（B）中分别肺炎链球菌的17株。每个符号代表CFU从一个大鼠的肺确定与代表组线平均值和标准差。量化（LLQ）的下限为1.7日志10 CFU /肺。化合物进行称重（56或112毫克的纯游离母体分子的），溶解在9毫升无菌水，稀释3毫升到3毫升水（1:1）和每日两次1毫升/125克大鼠经口管饲施用（ 6 - 7小时间隔）2天，开始后1小时的感染。未处理对照（NTC）的在感染后对于基线1小时进行采样或用盐水治疗和取样在研究（48小时）生长控制的结束。 从生物有机与药物化学快报，第21卷，TJ Miles 等，新颖的环己基酰胺转载作为强有力的抗菌药物靶向细菌IIA型拓扑异构酶图3A，页7483 - 7488，版权所有（2011）16，爱思唯尔的权限。从生物有机＆药物化学快报，第26卷，TJ Miles 等，新型三环类（ 例如 GSK945237）细菌IIA型拓扑异构酶的作为有效抑制剂，页2464重印图3B - 2469，版权所有（2016）17，与来自爱思唯尔许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55068/55068fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55068/55068fig4.jpg" /> 图4:PK /新型PDF抑制剂PD表征。 EMAX模型使用剂量范围在与肺炎链球菌 （A）或流感嗜血杆菌 （B）的菌株肺部感染的免疫活性小鼠的疗效数据，以确定一个新的PDF抑制剂18 PK / PD目标产生。界表示从与不同剂量的化合物处理4-5只小鼠/组的平均细菌负担。进行分析，以提供免费药物24小时AUC（空心圆）或AUC / MIC（实心圆）的疗效相关。授权转载，版权所有©美国微生物学会，[抗菌制剂和化疗，60卷，2016年，180页- 189] 18。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55068/55068fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td></td><td></td><td colspan="2"> 瘀 </td><td colspan="2"> 1 - 10日志减少 </td><td></td></tr><tr><td> 生物 </td><td> MIC （微克/毫升） </td><td> R 2的 线网络连接T（％） </td><td> fAUC </td><td> fAUC / MIC </td><td> fAUC </td><td> fAUC / MIC </td><td> 马克斯杀 （日志减少10） </td></tr><tr><td> 肺炎链球菌 </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 10127 </td><td> 0.25 </td><td> 99.9 </td><td> 2.0 </td><td> 8.1 </td><td> 3.6 </td><td> 14.4 </td><td> 2.8 </td></tr><tr><td> 1316009S </td><td> 0.25 </td><td> 96.1 </td><td> 6.5 </td><td> 26.0 </td><td> 15.1 </td><td> 60.3 </td><td> 1.8 </td></tr><tr><td> ATCC10813 </td><td> 0.5 </td><td> 100 </td><td> 8.4 </td><td> 16.9 </td><td> 22.2 </td><td> 44.5 </td><td> 1.2 </td></tr><tr><td> 1307007S </td><td> 1 </TD&gt; <td> 95.8 </td><td> 16.1 </td><td> 16.1 </td><td> 20.1 </td><td> 20.1 </td><td> 2.7 </td></tr><tr><td> ATCC6303 </td><td> 1 </td><td> 99.8 </td><td> 19.8 </td><td> 19.8 </td><td> 24.8 </td><td> 24.8 </td><td> 2.8 </td></tr><tr><td> 1629 </td><td> 2 </td><td> 100 </td><td> 15.8 </td><td> 7.9 </td><td> 21.2 </td><td> 10.6 </td><td> 2.4 </td></tr><tr><td> 298443 </td><td> 2 </td><td> 99.9 </td><td> 26.5 </td><td> 13.2 </td><td> 31.0 </td><td> 15.5 </td><td> 2.3 </td></tr><tr><td> 336808 </td><td> 2 </td><td> 86 </td><td> 8.2 </td><td> 4.2 </td><td> 19.0 </td><td> 9.5 </td><td> 2.6 </td></tr><tr><td> 338860 </td><td> 2 </td><td> 99.5 </td><td> 2.9 </td><td> 1.5 </td><td> 6.7 </td><td> 3.4 </td><td> 2.8 </td></tr><tr><td> ＆＃160; 340449 </td><td> 2 </td><td> 94.6 </td><td> 8 </td><td> 4 </td><td> 14.9 </td><td> 7.4 </td><td> 2.1 </td></tr><tr><td> L11259 </td><td> 2 </td><td> 100 </td><td> 7.1 </td><td> 3.5 </td><td> 9 </td><td> 4.5 </td><td> 2.0 </td></tr><tr><td>平均值±SD </td><td> 一个 NA </td><td> NA </td><td> 11.0±7.5 </td><td> 11.0±7.9 </td><td> 17.0±8.2 </td><td> 19.5±17.8 </td><td> 2.3±0.5 </td></tr><tr><td>中位数</td><td> NA </td><td> NA </td><td> 8.2 </td><td> 8.1 </td><td> 19.0 </td><td> 14.4 </td><td> 2.4 </td></tr><tr><td> 流感嗜血杆菌 </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 1998-100-126H </td><td> 1 </td><td> 99.7 </td><td> 7.3 </td><td> 7.3 </td><td> 13.4 </td><td> 13.4 </td><td> 2.9 </td></tr><tr><td> 503-008H </td><td> 1 </td><td> 99.8 </td><td> 7.1 </td><td> 7.1 </td><td> 13.9 </td><td> 13.9 </td><td> 2.7 </td></tr><tr><td> 08003H </td><td> 2 </td><td> 99.9 </td><td> 14.5 </td><td> 7.2 </td><td> 16.7 </td><td> 8.3 </td><td> 2.7 </td></tr><tr><td> H128 </td><td> 2 </td><td> 96.6 </td><td> 15.3 </td><td> 7.6 </td><td> 26.0 </td><td> 13.0 </td><td> 2.7 </td></tr><tr><td> 19001H </td><td> 4 </td><td> 91.9 </td><td> 14.8 </td><td> 3.7 </td><td> 37.3 </td><td> 9.3 </td><td> 3.0 </td></tr><tr><td>平均值±SD </td><td> NA </td><td> NA </td><td> 11.8±4.2 </td><td> 6.6±1.6 </td><td> 21.5±10.2 </td><td> 11.6±2.6 </td><td> 2.8±0.1 </td></tr><tr><td>中位数</td><td> NA </td><td> NA </td><td> 14.5 </td><td> 7.2 </td><td> 16.7 </td><td> 13.0 </td><td> 2.7 </td></tr><tr><td> 一个 NA，不适用。 </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表 1: 对 肺炎链球菌 和 流感嗜血杆菌 菌株 新型PDF抑制剂PK / PD目标 。自由药物的AUC和瘀AUC / MIC目标和一个1-登录从基线CFU 10减少在肺感染并用化合物18处理的免疫活性小鼠，测定了11 肺炎链球菌和5 流感嗜血杆菌 。该DAT一个被用来帮助人类选择剂量的临床开发。该表已被改编并许可转载，版权所有©美国微生物学会，[抗菌制剂和化疗，60卷，2016年，180页- 189] 18。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> 治疗一组 </td><td colspan="8"> 平均对10 肺炎链球菌 （CFU /肺）b±SD应变（AMX / CA MIC）: </td></tr><tr><td> 05010S （2微克/毫升） </td><td> 16001Sç （4微克/毫升） </td><td> 16001S （4微克/毫升） </td><td> 30005S （4微克/毫升） </td><td> 20009S （4微克/毫升） </td><td> 05003S （8微克/毫升） </td><td> 404053 （8微克/毫升） </td><td> 47003S （8微克/毫升） </td></tr><tr><td> 控制 </str翁&gt; </td><td> 7.3±0.4 </td><td> 7.0±0.8 </td><td> 5.7±1.2 </td><td> 7.1±0.7 </td><td> 7.1±0.4 </td><td> 6.4±0.6 </td><td> 6.8±0.4 </td><td> 6.0±0.3 </td></tr><tr><td> AMX / CA </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2000/125毫克的出价（比例16:1） </td><td> ≤1.7 ** </td><td> 2.8±1.2 ** </td><td> 2.2±0.8 ** </td><td> 2.3±0.9 ** </td><td> 2.5±0.9 ** </td><td> 2.0±0.9 ** </td><td> 3.8±1.4 ** </td><td> 1.8±0.2 ** </td></tr><tr><td> 1000/125毫克TID（比率8:1） </td><td> NT </td><td> NT </td><td> 2.8±0.5 ** </td><td> 2.7±1.3 ** </td><td> 3.3±0.9 ** </td><td> 4.9±0.5 ** </td><td> 6.6±1.3 </td><td> 4.7±0.7 ** </td></tr><tr> &lt;TD&gt; 875/125毫克TID（比率7:1） </td><td> NT </td><td> NT </td><td> 3.0±1.3 * </td><td> 2.1±0.7 ** </td><td> 3.2±0.4 ** </td><td> 5.2±0.9 * </td><td> 6.4±0.6 </td><td> 4.9±0.4 ** </td></tr><tr><td> 875/125 mg，每天两次（比7:1） </td><td> NT </td><td> NT </td><td> 4.5±1.2 * </td><td> 5.6±1.3 * </td><td> 5.2±0.7 ** </td><td> 6.3±0.7 </td><td> 6.4±0.7 </td><td> 5.5±0.4 </td></tr><tr><td> 500/125毫克TID（比率4:1） </td><td> 3.1±1.3 ** </td><td> 4.5±0.9 ** </td><td> NT </td><td> NT </td><td> NT </td><td> NT </td><td> NT </td><td> NT </td></tr><tr><td>阿奇霉素，1000/500毫克OD </td><td> NT </td><td> NT </td><td> NT </td><td> 5.8±2.0 </td><td> 7.1±0.9 </td><td> 3.1±1.0 ** </td><td> 6.6±0.4 </td><td> 67; 0.6 </td></tr><tr><td>左氧氟沙星，500毫克OD </td><td> NT </td><td> NT </td><td> 4.2±0.8 * </td><td> 5.7±0.8 * </td><td> 4.9±0.8 ** </td><td> 3.0±1.3 ** </td><td> 3.5±0.2 ** </td><td> 3.9±0.8 ** </td></tr><tr><td rowspan="2"> 治疗d组 </td><td colspan="8"> 平均登录10 流感嗜血杆菌 （CFU /肺）±SD应变: </td></tr><tr><td colspan="4"> H128 （β内酰胺酶阳性） </td><td colspan="4"> 切斯特菲尔德 （β内酰胺酶阴性，氨苄青霉素抗性） </td></tr><tr><td> 控制 </td><td colspan="4"> 6.4±0.6 </td><td colspan="4"> 6.7±0.6 </td></tr><tr><td> AMX / CA </td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>2000/125毫克的出价（比例16:1） </td><td colspan="4"> 2.0±0.7 ** </td><td colspan="4"> 3.1±0.9 ** </td></tr><tr><td> 1000/125毫克TID（比率8:1） </td><td colspan="4"> 2.1±1.0 ** </td><td colspan="4"> 3.6±1.1 ** </td></tr><tr><td> 875/125毫克TID（比率7:1） </td><td colspan="4"> 2.8±1.3 ** </td><td colspan="4"> 3.8±1.3 ** </td></tr><tr><td> 875/125 mg，每天两次（比7:1） </td><td colspan="4"> 2.0±0.8 ** </td><td colspan="4"> 5.1±1.1 ** </td></tr><tr><td>阿奇霉素，1000/500毫克OD </td><td colspan="4"> 3.2±1.7 * </td><td colspan="4"> 5.8±1.1 </td></tr><tr><td>左氧氟沙星，500毫克OD </td><td colspan="4"> ≤1.7 ** </td><td colspan="4"> ≤1.7 ** </td></tr><tr><td colspan="9"> 一个 AMX / CA，阿莫西林-克拉维酸;出价，每天两次; TID。， 一天三次;外径，每天一次。 </td></tr><tr><td colspan="9"> -B检测限为&lt;1.7; *，从控制显著差异（P≤0.05）; **从控制显著差异（P≤0.01）; NT，未经测试。 </td></tr><tr><td colspan="9"> C菌株16001S在两个独立的实验中进行测试。 </td></tr><tr><td colspan="9"> ð阿莫西林-克拉维酸500/125毫克（4:1）每日三次没有对流感嗜血杆菌菌株进行测试。 </td></tr></tbody></table> 表2: 重建人体血浆暴露型材不同阿莫西林/克拉维给药方案的疗效。在使用这个模型肺炎链球菌或流感嗜血杆菌分离株的感染肺免疫大鼠产生的数据显示，2阿莫西林-克拉维酸的增强型制剂（，000/125毫克，每天两次）比对以高标准菌株的给药方案体外药敏19更有效。该表已被改编并许可转载，版权所有©美国微生物学会，[抗菌制剂和化疗，音量49，2005年，908页- 915] 19。

Watch this Scientific Journal Video about A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

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Immunology and Infection

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

14.5K 次观看.  GlaxoSmithKline. A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

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Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

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 itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

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 ...

科研

免疫与感染

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

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

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

A Precise Pathogen Delivery and Recovery System for Murine Models of Secondary Bacterial Pneumonia

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, murine models of co-infection have been the primary tool developed to explore the pathologies of both the primary and secondary infections. Despite the prevalence of this model, considerable discrepancies regarding instillation procedures, dose volumes, and efficacies are prevalent among studies. Furthermore, these efforts have been largely incomplete in addressing how the pathogen may be directly influencing disease progression post-infection. Herein we provide a precise method of pathogen delivery, recovery, and analysis to be used in murine models of secondary bacterial pneumonia. We demonstrate that intratracheal instillation enables an efficient and accurate delivery of controlled volumes directly and evenly into the lower respiratory tract. Lungs can be excised to recover and quantify the pathogen burden. Following excision of the infected lungs, we describe a method to extract high quality pathogen RNA for subsequent transcriptional analysis. This procedure benefits from being a non-surgical method of delivery without the use of specialized laboratory equipment and provides a reproducible strategy to investigate pathogen contributions to secondary bacterial pneumonia.

Here, we present methods to improve secondary bacterial pneumonia studies by providing a non-invasive route of instillation into the lower respiratory tract followed by pathogen recovery and transcript analysis. These procedures are reproducible and can be performed without specialized equipment such as cannulas, guide wires, or fiber optic cables.

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

Secondary bacterial pneumonias following influenza infections consistently rank within the top ten leading causes of death in the United States. To date, ...

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 &#181;L of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative ...

医学

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

病毒合并感染后 肺炎链球菌 从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

评估肺炎消退的实验模型

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

Preparation of Agar Bead Embedded Mycobacterium abscessus to Inoculate Immunocompetent Mice Intratracheally

Robust mouse models of chronic infection with Mycobacterium abscessus, an environmental pathogen that preferably infects the lungs, are badly needed. Year-long multidrug therapies deliver unacceptably poor cure rates in patients, around 50%, hence the need for predictive preclinical tools to develop better antibiotics. However, immunocompetent mice are generally resistant to pulmonary infection by M. abscessus. Numerous attempts to establish a sustained infection with M. abscessus in immunocompetent mice have been reported in the literature. Among these, methods relying on agar bead-embedded bacteria inoculated into C57BL/6 mice via the intratracheal route have proven most promising. The major limitation of this approach is the technical challenge associated with the preparation of agar beads of reproducible size and bacterial numbers, followed by intratracheal inoculation. Here, we first provide a detailed description of an optimized protocol that delivers M. abscessus-loaded agar beads of optimal diameter and reproducible bacterial burden. Next, we provide a detailed protocol of intratracheal inoculation, optimized to avoid losses of beads and bacteria due to adherence to surfaces of the inoculation devices and to achieve reproducible pulmonary deposition of agar-embedded M. abscessus. The objective is to ensure ease of implementation and excellent lab-to-lab reproducibility.

Mice are generally resistant to infections by Mycobacterium abscessus, which complicates the discovery and development of much-needed antibiotics against pulmonary infections. Here, we describe a method of inoculum preparation and intratracheal infection that was shown to deliver sustained infection in immunocompetent mice.

Robust mouse models of chronic infection with Mycobacterium abscessus, an environmental pathogen that preferably infects the lungs, are badly needed. ...

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对

摘要

探索更多视频

此视频中的章节

长期慢性

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

绿脓杆菌引起的肺损伤模型

研究微生物群落

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型

病毒合并感染后肺炎链球菌从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

评估肺炎消退的实验模型

Preparation of Agar Bead Embedded Mycobacterium abscessus to Inoculate Immunocompetent Mice Intratracheally

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对

摘要

探索更多视频

此视频中的章节

长期慢性

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

绿脓杆菌引起的肺损伤模型

研究微生物群落

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

二次细菌性肺炎的鼠种模型的精确病原体输送与恢复系统

呼吸机相关和医院获得性肺炎的小鼠口咽吸入模型

病毒合并感染后 肺炎链球菌 从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

评估肺炎消退的实验模型

Preparation of Agar Bead Embedded Mycobacterium abscessus to Inoculate Immunocompetent Mice Intratracheally

病毒合并感染后肺炎链球菌从定植者转变为病原体的小鼠模型概括了年龄加重的疾病