This work was supported in part by the Center for Integration of Medicine and Innovative Technology (CIMIT) under the U.S. Army Medical Research Acquisition Activity Cooperative Agreement (CIMIT No. 14-1894 to TD) and the National Institutes of Health (1R21AI109172 to TD). YW was supported by an ASLMS Student Research Grant (BS.S02.15). We are grateful to Tayyaba Hasan, PhD at the Wellman Center for her co-mentorship for YW.

1.细菌培养的制备<ol><li>添加7.5毫升脑心浸液（BHI）培养基中至50mL离心管中。种子鲍曼不动杆菌细胞在BHI培养基，然后孵育鲍曼不动杆菌培养在轨道培养箱（37℃）18小时。 </li><li>离心细胞的培养物在3,500×g下5分钟，除去上清液，并在磷酸盐缓冲盐水（PBS）洗涤小球。 </li><li>重悬在新鲜的PBS中的细菌颗粒和彻底吸管的悬浮液。 </li><li>收集100μL的细菌悬浮液，并使用新鲜的PBS作1:10稀释。 </li><li>转移稀释到1.5mL半微量比色杯，并测量在600nm（OD 600 nm）的波长的光密度（OD）。 </li><li>根据稀释的测量OD 600nm的值与稀释倍数（10）计算原始（未稀释的）悬浮液的OD 600纳米在PBS中。 </li><li>调整日在PBSë原始悬浮液至OD 600纳米 = 0.6（对应于10 8 CFU / mL的细胞密度）。 </li></ol>通过生物发光甲导致了烧伤感染的2小鼠模型。 鲍曼不动杆菌 <ol><li>年龄7-8周，体重17〜19克使用成年雌性BALB / c小鼠。允许小鼠实验开始前适应环境实验室条件至少3天。保持在12小时光照/黑暗周期的小鼠的21℃室温下，给他们的食物和水随意 。 </li><li>麻醉小鼠用氯胺酮 - 赛拉嗪混合物的腹膜内注射（100mg / kg的 - 20毫克/千克）。轻轻触摸每只小鼠用棉签的睑;不存在所述眼睑反射的暗示适当的麻醉深度。盖鼠标眼睛兽医药膏，以防止干燥眼睛在麻醉过程中。 </li><li>剃须背面的小鼠用5揭露尽可能多的皮肤尽可能0刀理发器。 </li><li>放置在35毫米培养皿中的小鼠的腹部下方的盖，以保持他们的背部在相对水平的位置。 </li><li>使用10&quot; ×10&quot; ，220 VAC热板在250毫升烧杯（80％满）烧开水。直到达到与水热平衡浸入黄铜块（1 1cm×1cm的横截面）放入烧杯中。热平衡通常需要&lt;5分钟，并通过在烧杯中的水的再沸腾指示。 </li><li>在此之前创建烧伤，辖先发制人镇痛药（为0.1mg / kg的丁丙诺啡的皮下注射），用于缓解疼痛。 </li><li>先发制人镇痛药10分钟后，轻轻按下加热黄铜块对小鼠为7秒背面的剃毛区以诱导烧伤创面。 注:为避免热损伤到工作人员，在执行烧录程序时戴上热防护手套。 </li><li>管理通过subcutaneou 0.5毫升的无菌盐水的注射液，以防止脱水。 </li><li>五分钟后的热损伤的诱导，接种含有5×10 6 CFU的PBS细菌悬浮液的50微升到使用移液管的小鼠烫伤。通过在皮肤上的锯齿形运动移动无菌棉签涂抹在灼伤等分在过火面积尽可能均匀地分布在细菌细胞。 </li><li>细菌接种后，立即进行生物发光成像的感染的烧伤，如第4说明。 </li><li>放置小鼠上的水加热外科床（37℃，回收区），直到小鼠已经完全从麻醉中恢复。房子与在生物安全等级-2动物室的每笼5只的最大数量的小鼠。 </li><li>施用镇痛药（0.1mg / kg的丁丙诺啡的皮下注射），每天两次对烧伤后的前三天。 </li></ol> 3.抗菌蓝色光疗法对于A＆＃160; 杆菌感染小鼠<ol><li>在细菌接种后24个小时开始ABL治疗。 </li><li>使用的发光二极管（LED），其具有用于照射ABL 415nm处的峰值发射。安装在一个散热片上的LED，以防止对小鼠18所照射的区域的热效应。固定LED与夹子连接器的光学支承杆，以允许LED以向上和向下移动。 </li><li>打开电源/能量计和按波长按钮选择415纳米。重置电源/能量计减去背景（环境光）。 </li><li>将电源/电能表右侧的LED下。穿蓝色光护目镜。开启LED光并调整LED孔（即来自LED会聚的光的透镜）和功率/能量计的光传感器（2厘米直径）之间的距离，使得光斑覆盖的整个区域光传感器。 </li><li>仔细调节LED驱动器和记录鲍威的阅读R /电能表。根据读取计算的辐照度:辐照度=读数（W）/面积（cm 2）。通过调整LED驱动器调节LED 100毫瓦/厘米2的辐照度。 </li><li>关闭LED。测量LED孔和功率/能量计的光传感器之间的距离。 </li><li>麻醉小鼠用氯胺酮 - 赛拉嗪混合物的腹膜内注射（100mg / kg的 - 20毫克/千克）。不存在所述眼睑反射的暗示适当的麻醉深度。 </li><li>随机划分小鼠成ABL治疗组（n = 10）和未处理的对照组（n = 10）。 </li><li>对于ABL处理组，覆盖小鼠的眼睛用铝箔以避免过度暴露于光。将鼠标烧伤直属LED，用鼠标腹部下方的35毫米陪替氏培养皿的盖子，以保持他们的背部在水平位置。 </li><li>在一个正方形皮氏二取代，在步骤3.5，用鼠标提到的功率/能量计SH。调整鼠标回的位置处，如步骤所讨论的LED孔和鼠标烧伤的表面之间的距离是等于LED孔和电源/能量计的光传感器（水平之间的高度3.5）。 </li><li>以100mW / cm 2的辐照度照射感染的烧伤。递送ABL在剂量72焦耳/厘米2，直到360达到焦耳/ cm 2的总剂量（ 例如 ，0，72，144，216，288和360焦耳/厘米2）。各光剂量后，执行生物发光成像的小鼠，如第4所讨论的。 </li><li>对于未处理的对照组，执行鼠标烧伤生物发光成像，如第4所讨论的，使用如用于ABL处理组相同的时间间隔。 </li><li>小鼠的恢复之前，放置在水加热手术床上的小鼠以防止热量散失和低温。观察活动和老鼠，直到复苏的眼睑反射。后日小鼠电子回收，安置他们2-3个笼子。 </li><li> ABL治疗后，执行生物发光成像，如第4对第3天所讨论的，每日然后隔天以监测感染的小鼠的时间生物负担。 </li></ol> 4.感染的生物发光成像小鼠<ol><li>图像使用低光成像系统，其包括增强型电荷耦合器件照相机，摄像机控制器，一个样品室，以及图像处理器19中的小鼠。 </li><li>麻醉小鼠用氯胺酮 - 赛拉嗪混合物的腹膜内注射（100mg / kg的 - 20毫克/千克）。轻触用棉签小鼠的眼睑;不存在所述眼睑反射的暗示适当的麻醉深度。 </li><li>启动实时成像软件。在显示的控制面板，单击初始化。等到温度框的颜色变为绿色，表明阶段的温度在试样腔室已经达到了37℃。 </li><li>放置在成像系统的样本腔室中的阶段（37℃）的小鼠中，与直接在相机下的感染的烧伤。 注:当烧伤变得干燥细菌的生物发光可以降低。因此，建议在成像之前润泽用PBS小鼠烧伤。 </li><li>在控制面板中，把一个对号旁边的"发光"。选择"自动曝光"，使成像的曝光时间将通过基于生物发光强度的活体成像软件进行优化。 </li><li>从"查看现场的"下拉列表中选择"C"。选择"扫描中间范围"选项，让软件判断焦距。将一个复选标记旁边为叠加。 </li><li>点击"获取"来捕捉图像。在"编辑图像标签"框中，单击"确定"; "图像窗口"和"收费调色板"将出现。 </li><li>设置自动选择自动ROI参数。 </li><li>量化生物发光强度为相对发光单位（的RLU）和伪彩色尺度从粉红（最强烈）到蓝色（至少激烈）19，20显示生物发光。 </li><li>计算在不同的基于生物发光强度分析的时间点在小鼠烧伤细菌的存活分数。在给定时间点的细菌的存活分数=在该时间点测量到的生物发光强度/生物发光强度ABL曝光17前右测量。 </li></ol> 5.小鼠安乐死<ol><li>是该实验的终点为如下:（a）如由生物发光损失作为通过成像确定的感染的分辨率; （B）系统的感染的发生通过生物发光的传播烧成以外如所指示一个;在比较的≥15％体重（c）中损失到正常年龄匹配的小鼠中，或从疼痛和痛苦由缺乏响应于手动刺激，不动，一个不能吃或喝所指示的痛苦。在研究过程中，如果我们遇到这样的情况，我们不确定鼠标是否已经完全达到了奄奄一息的状态，根据我们的定义所概述，和/或我们是不确定的，如果我们需要安乐死它的情况，我们会联系CCM兽医人员行动计划; （d）中，否则将小鼠研究开始后30天被安乐死。 </li><li> 重新定位到小鼠关闭笼子专门为安乐死和通过递送二氧化碳安乐死的小鼠（CO 2）的压缩气体进入笼中。 <ol><li>打开CO 2罐或稳压阀以启动气体的流动。验证读取调节器根据刊登的单元的指令正确的PSI（磅每平方英寸），并调整重新根据需要gulator到正确的PSI，通常不高于5磅。 </li><li>慢慢填写。流速应置换每分钟腔室/笼体积的不超过30％（对于典型的鼠笼，〜2L /分钟;对于鼠笼，〜7.5升/分钟）。 </li><li>等待约3-5分钟为动物停止移动或呼吸;眼睛应该是固定和扩张。关闭CO 2罐或调节阀，停止CO 2的流动。 </li><li>确保心脏没有摸着拇指和食指之间的胸口跳动。确保有通过触摸眼球没有眨眼反射。 <ol><li>如果有一个心跳或眨眼反射，重复安乐死过程或使用剪刀来打开胸腔以创建气胸（动物必须是非响应于脚趾捏之前执行此过程）。 </li></ol></li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individualised+antibiotic+dosing+for+patients+who+are+critically+ill:+challenges+and+potential+solutions.">Individualised antibiotic dosing for patients who are critically ill: challenges and potential solutions.</a> Lancet Infect Dis. 14 (6), 498-509 (2014).</li><li>Dai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+light+eliminates+community-acquired+methicillin-resistant+Staphylococcus+aureus+in+infected+mouse+skin+abrasions.">Blue light eliminates community-acquired methicillin-resistant Staphylococcus aureus in infected mouse skin abrasions.</a> Photomed Laser Surg. 31 (11), 531-538 (2013).</li><li>Uppu, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+side+chain+amphiphilic+polymers+disrupt+surface+established+bacterial+bio-films+and+protect+mice+from+chronic+Acinetobacter+baumannii+infection.">Amide side chain amphiphilic polymers disrupt surface established bacterial bio-films and protect mice from chronic Acinetobacter baumannii infection.</a> Biomaterials. 74, 131-143 (2016).</li><li>Donlan, R. M., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+survival+mechanisms+of+clinically+relevant+microorganisms.">Biofilms: survival mechanisms of clinically relevant microorganisms.</a> Clin Microbiol Rev. 15 (2), 167-193 (2002).</li><li>Olsen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+tolerance+and+resistance.">Biofilm-specific antibiotic tolerance and resistance.</a> Eur J Clin Microbiol Infect Dis. , (2015).</li><li>Song, H. 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The authors declare that they have no competing financial interests.

ABL是用于治疗感染的新方法。由于其作用机制不同于化疗的完全不同，它更是一个理疗。介导抗微生物效果的药剂是蓝色光照射（400-470纳米）。随着蓝光LED的发展，我们获得了进入的MDR感染的有效和简单的基于光抗菌方法。 在这个协议中，我们描述了造成MDR， 鲍曼不动杆菌的生物发光应变烧伤感染的小鼠模型的发展。与使用生物发光的细菌，感染的程度可以是非侵入性地实时监控在通过生物发光成像活体动物。利用细菌的生物发光工程化菌株和低光成像技术的创建用于抗菌治疗期间监控实时的感染的有效技术。这种方法也可以在感染的调查中使用由其他微生物物种以及位于其他网站。除了抗菌方法的疗效评估，这种方法也可以用来跟踪感染的进展。 通过使用该小鼠模型中，我们表明，ABL（415 nm）的成功灭活中建立感染（ 图1A和C）的细菌。之前ABL疗法，在已建立的感染（ 图1B），这是生物膜的特征，观察到细菌的簇。生物膜是更宽容的传统抗生素和宿主防御相比，他们的浮游6,7和经常与持续性感染8，9关联。代表结果在415纳米ABL看好的是生物膜渗透。此外，再加上以前的报告29，30，31，32，我们的结果表明，不管细菌的耐药性轮廓的ABL的有效性仍然存在。 这里描述的协议涉及三个主要步骤:（1）烧伤感染的小鼠模型的发展，（2）ABL疗法，和（3）生物发光成像。在开发烧伤感染的小鼠模型中，我们注意到，有迹象表明影响感染的程度和ABL的后续有效性几个因素:（1）燃烧时间会影响伤口的深度和细菌的增殖。当燃烧时间从3到7秒增加，细菌发光强得多（指示感染的较高程度）在24小时后接种和感染的消除需要高得多的ABL曝光（&gt; 360焦耳/ cm 2的）。 （2）细菌的接种物是对于开发的关键参数˚F感染。较高的细菌接种物通常会导致更高的感染程度，而具有足够低的接种物经常未能开发小鼠稳定感染。在后一种情况下，细菌发光通常会成为细菌接种后不久就检测不到。 （3）细菌和宿主之间的相互作用依赖于细菌种类。我们还用铜绿假单胞菌建立一个感染模型。我们发现，在同等条件下（ 即，燃烧时间和细菌接种），由铜绿假单胞菌引起的感染的进展速度远远超过鲍曼不动杆菌感染，以及败血症总是在小鼠体内48小时接种后25内观察到。 对于ABL治疗的执行，存在需要解决的几个重要的要点:（1）正确光辐照所需的ABL疗法的功效最大化。 （2）燃烧的所述的表面小鼠小号HOULD被作为水平放置越好。故障的烧伤表面适当地定位可影响ABL治疗的功效。 （3）在光曝光，故建议小鼠的眼睛用铝箔进行保护，特别是当使用激光作为光源。 （4）在曝光，应小心监测情况下，它们从麻醉中苏醒的小鼠。在这种情况下，麻醉药的一小笔额外剂量应给予以保持麻醉动物。 （5）两个ABL处理小鼠和未处理小鼠的应放置在加热床下麻醉时，以维持体温。在生物发光成像的过程中，当燃烧变得干燥细菌的生物发光可降低。因此，建议在成像之前润泽用PBS小鼠烧伤。 还有在这个协议所讨论的技术的一些限制:（1）对于EXTE的监测目的感染实时的NT，必须使用生物发光细菌菌株。因此，前一个临床菌株可在动物模型中进行测试，就必须进行遗传由勒克斯CDABE操纵子11的转染改性。 （2）ABL的有效性相关的波长33和细菌物种/株使用34。的蓝光波长，与其它参数一起，应进一步用于灭活不同细菌物种/株进行了优化。 （3）我们只研究了小鼠浅表感染。对于深层感染，ABL的局部递送可能无法到达感染，所以可能需要35间质性光递送。 （4）是成像系统的灵敏度限制，成像深部感染19时尤其如此。其结果是，即使当生物发光的像素被完全消除，有可能仍然是六能细菌细胞剩余，允许发生细菌再生。一个长时间暴露于ABL是消除细菌发光的建议后，为了防止细菌再生。

烧伤感染，这是因为皮肤的热损伤的频传，继续成为发病率和死亡率1的一个重要原因。烧伤感染的管理已经通过多药耐药性（MDR）的细菌菌株2的增加出现了进一步的损害，由于大量使用抗生素。一个重要的MDR革兰阴性菌是鲍曼不动杆菌 ，这是已知的与最近的战斗创伤相关联且几乎所有可用的抗生素耐药3。生物膜的损伤灶的存在已经报告了4，5，被认为是加剧耐受抗生素和宿主防御6，7，引起持续感染的8，9。因此，有一种压力机，加工摹需要替代治疗的发展。在最近公布的国家战略打击抗生素耐药细菌 ，替代疗法，抗生素的发展已注意到由美国10政府的作用。 光为基础的抗菌剂的方法，由名称所指示的，需要与或不与其它药剂的光照射。这些方法包括抗微生物光动力学疗法（APDT），紫外线C（UVC）照射，和抗微生物蓝光（ABL）。在以前的研究中，他们已经杀死MDR菌株11，12，13显示出可喜的成效。在这三种光为基础的方法，ABL已吸引近几年越来越多的关注，由于无需使用光敏剂14的其固有的抗菌性能。在COMPARISON到APDT，ABL仅涉及使用光的，而需要APDT光和光敏剂的组合。因此，ABL是简单且廉价的14。相比于UVC，ABL被认为是细胞毒性和遗传毒性对宿主细胞15要少得多。 此协议的目的是研究ABL对小鼠模型引起MDR 鲍曼不动杆菌烧伤感染的治疗功效。我们用生物发光致病细菌培养烧伤感染，允许的实时细菌负荷的无创监测的新的小鼠模型。相比，体液/组织提取和随后的镀敷和菌落计数16传统的方法，这种技术提供了精确的结果。组织取样的过程中可能引入的实验误差的另一个来源。由于细菌的发光强度成线性比例的受文者积水细菌CFU 17，我们可以直接测量光照射一定剂量后的细菌的存活。通过监测在活的动物中实时接收光处理中的细菌负荷，的细菌杀死动力学可使用小鼠的显著减少数量来表征。

<table><tbody><tr><td>IVIS&amp;nbsp;</td><td>PerkinElmer Inc, Waltham, MA</td><td>IVIS Lumina Series III</td><td>Pre-clinical &lt;em&gt;in vivo&lt;/em&gt; imaging</td></tr><tr><td>Light-emitting diode LED</td><td>VieLight Inc, Toronto, Canada&amp;nbsp;</td><td>415 nm</td><td>Light source for illumination</td></tr><tr><td>Power/energy meter</td><td>Thorlabs, Inc., Newton, NJ</td><td>PM100D</td><td>Light irradiance detector</td></tr><tr><td>Mouse&amp;nbsp;</td><td>Charles River Laboratories, Wilmington, MA</td><td>BALB/c</td><td>7-8 weeks age, 17-19 g weight</td></tr><tr><td>&lt;em&gt;Acinetobacter baumannii&amp;nbsp;&lt;/em&gt;</td><td>Brooke Army Medical Center, Fort Sam Houston, TX</td><td>Clinical isolate</td><td>Engineered luminescent strain</td></tr><tr><td>Insulin Syringes</td><td>Fisher Scientific</td><td>14-826-79</td><td>BD Lo-Dose U-100 Insulin Syringes for injection</td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>721016</td><td>0.9% Sodium Chloride</td></tr><tr><td>Phosphate Buffered Saline, 1x Solution</td><td>Fisher Scientific</td><td>BP24384&amp;nbsp;</td><td>A standard phosphate buffer used in many biomolecular procedures</td></tr><tr><td>Brain Heart Infusion</td><td>Fisher Scientific</td><td>B11059</td><td>Bacterial culture medium</td></tr><tr><td>Falcon 15 mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-959-70C</td><td>For bacterial suspension centrifuge</td></tr><tr><td>Benchtop Incubated Orbital Shakers</td><td>Laboratory Supply Network, Inc, Atkinson, NH&amp;nbsp;</td><td>Incu-Shaker Mini</td><td>For culturing of bacteria</td></tr><tr><td>Inoculating Loops</td><td>Fisher Scientific</td><td>22-363-605&amp;nbsp;&amp;nbsp;</td><td>For smearing bacterial inoclum on burn surface of mice</td></tr><tr><td>Fisher Scientific Redi-Tip Pipet Tips, 1-200 &amp;micro;L</td><td>Fisher Scientific</td><td>02-707-502</td><td>Pipet Tips</td></tr><tr><td>Thermo Scientific&amp;nbsp;Sorvall Legend X1 Centrifuge</td><td>Fisher Scientific</td><td>75-004-220</td><td>For bacterial suspension seperation</td></tr><tr><td>Brass Block</td><td>Small Parts, Inc., Miami, FL</td><td>10 mm by 10 mm&amp;nbsp;</td><td>For creation of burns in mice</td></tr><tr><td>Extreme Dragon PBI/Kevlar High-Heat Gloves</td><td>Superior Glove Works Ltd, Cheektowaga, NY</td><td>PBI83514&amp;nbsp;</td><td>Heat Resistant Gloves</td></tr><tr><td>Greiner dishes</td><td>Sigma-Aldrich Co. LLC</td><td>P5112-740EA</td><td>35 mm &amp;times;10 mm</td></tr><tr><td>Corning Digital Hot Plate</td><td>Cole-Parmer Instrument Company, LLC</td><td>UX-84301-65</td><td>10&quot; x 10&quot;, 220 VAC, for boiling water&amp;nbsp;</td></tr><tr><td>Mouse/Rat Thin Line Water Heated Surgical Bed</td><td>E-Z Systems</td><td>EZ-211</td><td>Prevents heat loss and hypothermia during surgery</td></tr><tr><td>Vet ointment</td><td>S&amp;#38;K Pharma, GmbH</td><td>Kerato Biciron 5%, Augensalbe</td><td>opthalmic ointment to prevent eye dryness</td></tr></tbody></table>

in vivo investigation of antimicrobial blue light therapy for multidrug-resistant acinetobacter baumannii burn infections using bioluminescence imaging

烧伤感染仍是发病率和死亡率的重要原因。多药耐药（MDR）细菌的日益兴起，导致了传统抗生素治疗的频繁故障。替代治疗迫切需要解决MDR细菌。 一个创新的非抗生素的办法，抗菌蓝光（ABL），已经显示出对MDR感染的有前途的有效性。 ABL的作用机制尚不清楚。它通常假设在细菌中天然存在的内源性光敏发色团（ 例如 ，无铁卟啉，黄素， 等 ）由ABL，这反过来又产生细胞毒性活性氧类（ROS）通过光化学方法激发。 不同于另一基于光的抗菌方法中，抗微生物光动力学疗法（APDT），ABL疗法不需要外源photosensitiz的参与呃。所有它需要生效是蓝色光的照射;因此，它是简单和廉价的。 ABL的受体是在细菌中内源性细胞的光敏剂，而不是DNA。因此，ABL被认为是少得多基因毒性对宿主细胞不是紫外线C（UVC）照射，这直接导致在宿主细胞中的DNA损伤。 在本文中，我们提出了一个协议，以评估ABL治疗的烧伤损伤的小鼠模型MDR 鲍曼不动杆菌感染的效果。通过使用生物发光的工程菌株，我们能够无创监测感染的实时程度在活的动物。这种技术也用于监测动物感染的空间分布的有效工具。

我们使用的是鲍曼不动杆菌菌株是MDR临床分离株，如先前12,17报道。该菌株是由luxCDABE歌剧11的转染由生物发光。 图1A示出了从代表性小鼠连续细菌发光图像烧伤感染5×10 6 鲍曼不动杆菌 ，并在细菌接种后24个小时暴露于单个ABL曝光。有代表性的小鼠皮肤的组织切片的革兰氏染色标本烧（在24小时后收获的接种）证明鲍曼不动杆菌生物膜的感染性烧伤（ 图1B）的表面上的存在。 如图1A所示，发光细菌几乎360焦耳/ cm 2的曝光ABL被递送（在照射60分钟后根除为100mW / cm 2）的辐照度。 图1C是从感染有5×10 6 鲍曼不动杆菌 ，并在24个小时细菌接种后（N = 10）用ABL处理的小鼠烧伤的平均细菌发光的剂量-反应曲线。为了实现3-登录小鼠烧伤10灭活鲍曼不动杆菌 ，约360焦耳/ cm 2的ABL是必需的。鼠标的细菌发光燃烧未曝光到ABL保持期间的时间的等效周期几乎保持不变（数据未显示; P &lt;0.001）。 <img alt="图1" src="/files/ftp_upload/54997/54997fig1.jpg" /> 图1:在受感染小鼠烧伤细菌的灭活ABL。 （A）从代表性小鼠逐次细菌发光图像烧伤感染鲍曼不动杆菌的5×10 6 CFU，并在24暴露于360焦耳/ cm 2的ABL细菌接种后小时。 （B）革兰氏染色的代表性小鼠皮肤烧伤表示在小鼠烧伤鲍曼不动杆菌生物膜（箭头）的存在下的组织切片。皮肤样品在细菌接种后24个小时收获细胞。 （C）小鼠的平均细菌发光的剂量-反应曲线烧伤感染5×10 6 鲍曼不动杆菌 ，并用在细菌接种后24个小时（N = 10）360焦耳/ cm 2的ABL的曝光。酒吧:标准偏差。 <a href="http://ecsource.jove.com/files/ftp_upload/54997/54997fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging at JoV

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

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by 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 ...

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Nanyang Technological University

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Immunology and Infection

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

9.2K 次观看.  Massachusetts General Hospital, Harvard Medical School. Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

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

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

在体内的双基体生物光学成像

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

科研

医学

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

免疫与感染

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

可见光和庆大霉素的协同作用<em&gt;假单胞菌假单胞菌</em&gt;微生物

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

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 ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

基于 LED 的体外 筛选用于评估细菌光动力灭活中的光激活分子

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

生物化学

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

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.
For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.
This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

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, ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

生物学

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

肿瘤进展和血管生成的双生物发光成像

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

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

摘要

探索更多视频

在体内的双基体生物光学成像

生物发光细菌成像

可见光和庆大霉素的协同作用

4D多模态成像

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

基于 LED 的体外筛选用于评估细菌光动力灭活中的光激活分子

使用生物发光成像对小鼠尿路感染及其治疗的纵向随访

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

肿瘤进展和血管生成的双生物发光成像

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

摘要

探索更多视频

在体内的双基体生物光学成像

生物发光细菌成像

可见光和庆大霉素的协同作用

4D多模态成像

白色念珠菌生物膜发展在医学上，国外相关机构在小鼠皮下型号其次是生物发光成像

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

基于 LED 的体外 筛选用于评估细菌光动力灭活中的光激活分子

使用生物发光成像对小鼠尿路感染及其治疗的纵向随访

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

肿瘤进展和血管生成的双生物发光成像

基于 LED 的体外筛选用于评估细菌光动力灭活中的光激活分子