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
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| IVIS | PerkinElmer Inc, Waltham, MA | IVIS Lumina Series III | Pre-clinical in vivo imaging |
| Light-emitting diode LED | VieLight Inc, Toronto, Canada | 415 nm | Light source for illumination |
| Power/energy meter | Thorlabs, Inc., Newton, NJ | PM100D | Light irradiance detector |
| Mouse | Charles River Laboratories, Wilmington, MA | BALB/c | 7-8 weeks age, 17-19 g weight |
| Acinetobacter baumannii | Brooke Army Medical Center, Fort Sam Houston, TX | Clinical isolate | Engineered luminescent strain |
| Insulin Syringes | Fisher Scientific | 14-826-79 | BD Lo-Dose U-100 Insulin Syringes for injection |
| Sodium Chloride | Fisher Scientific | 721016 | 0.9% Sodium Chloride |
| Phosphate Buffered Saline, 1X Solution | Fisher Scientific | BP24384 | A standard phosphate buffer used in many biomolecular procedures |
| Brain Heart Infusion | Fisher Scientific | B11059 | Bacterial culture medium |
| Falcon 15mL Conical Centrifuge Tubes | Fisher Scientific | 14-959-70C | For bacterial suspension centrifuge |
| Benchtop Incubated Orbital Shakers | Laboratory Supply Network, Inc, Atkinson, NH | Incu-Shaker Mini | For culturing of bacteria |
| Inoculating Loops | Fisher Scientific | 22-363-605 | For smearing bacterial inoclum on burn surface of mice |
| Fisher Scientific Redi-Tip Pipet Tips, 1-200µL | Fisher Scientific | 02-707-502 | Pipet Tips |
| Thermo Scientific Sorvall Legend X1 Centrifuge | Fisher Scientific | 75-004-220 | For bacterial suspension seperation |
| Brass Block | Small Parts, Inc., Miami, FL | 10 mm by 10 mm | For creation of burns in mice |
| Extreme Dragon PBI/Kevlar High-Heat Gloves | Superior Glove Works Ltd, Cheektowaga, NY | PBI83514 | Heat Resistant Gloves |
| Greiner dishes | Sigma-Aldrich Co. LLC | P5112-740EA | 35 mm ×10 mm |
| Corning Digital Hot Plate | Cole-Parmer Instrument Company, LLC | UX-84301-65 | 10" x 10", 220 VAC, for boiling water |
| Mouse/Rat Thin Line Water Heated Surgical Bed | E-Z Systems | EZ-211 | Prevents heat loss and hypothermia during surgery |
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