T4 Optically detectable antimicrobial peptides enable the immediate detection of bacteria and fungi in the lung
T4 Optically detectable antimicrobial peptides enable the immediate detection of bacteria and fungi in the lung
复制标题
T4 光学可检测抗菌肽能够立即检测肺部细菌和真菌
DOI：
10.1136/thoraxjnl-2015-207770.4
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发表时间：
2015
期刊：
影响因子：
10
通讯作者：
Akram A
中科院分区：
文献类型：
--
作者：
Akram A
简介立即检测重症监护病房不明原因肺部混浊患者肺部的病原体将代表其治疗的重大进步。光学成像策略，包括支气管内施用细菌特异性智能探针，将比支气管肺泡灌洗等传统技术具有许多优势，主要是实时检测以立即通知抗菌治疗。本研究的目的是荧光标记和迭代开发抗菌肽，使用纤维共聚焦荧光显微镜 (FCFM) 对肺部原位细菌进行成像。方法抗菌肽 (AMP) 在树状支架 (AMP-1) 上合成，并与称为 NBD 的环境敏感荧光团缀合，随后不断开发线性对应物。进一步的构建体由与 NBD 荧光团 (AMP-2) 缀合的具有革兰氏选择性的 AMP 组成。这些与 FCFM 相结合，可在细菌感染的离体羊模型中以微米分辨率进行远端肺泡成像。结果 AMP-1 以浓度依赖性方式显示细菌结合亲和力，并标记不同的细菌组，包括由 > 70% 的呼吸机相关性肺炎致病微生物和病原真菌烟曲霉组成的组。AMP-1 表现出明显高于大肠杆菌的等摩尔线性当量的荧光。大肠杆菌、肺炎克雷伯菌、铜绿假单胞菌、MSSA、A。鲍曼氏菌和 S.肺炎链球菌（所有 p < 0.01）对细菌的选择性高于对哺乳动物细胞的选择性，并且与急性呼吸窘迫综合征患者的支气管肺泡灌洗液一起孵育时，其化学稳定性优于线性等效物。此外，AMP-1可以标记E。大肠杆菌、肺炎克雷伯菌、铜绿假单胞菌和 MSSA 经支气管内滴注并用 FCFM 成像时的原位活体动物模型（针体外，相对于哺乳动物细胞，对革兰氏阴性菌保持选择性。在离体模型中，AMP-2 选择性标记革兰氏阴性菌片段（铜绿假单胞菌、肺炎克雷伯菌和大肠杆菌）而不是革兰氏阳性菌片段（MSSA、MRSA 和肺炎链球菌）或控制肺段（所有 p < 0.05）。 结论 描述了一种在大小相关的临床前模型中立即检测具有克选择性的细菌的 Smartprobe/FCFM 策略，并且正在进行首次人体转化。
IntroductionThe immediate detection of pathogens in the lungs of patients with unexplained pulmonary opacities in the intensive care unit would represent a significant advance in their management. An optical imaging strategy, including the endobronchial administration of bacterial specific Smartprobes, would confer a number of advantages over conventional techniques such as bronchoalveolar lavage, principally real-time detection to immediately inform antimicrobial therapy. The aims of this study were to fluorescently label and iteratively develop anti-microbial peptides to image bacteriain situin the lung using fibered confocal fluorescence microscopy (FCFM).MethodsAntimicrobial peptides (AMP) have been synthesised on a dendrimeric scaffold (AMP-1) and conjugated to an environmentally sensitive fluorophore called NBD, following the continuous development a linear counterpart. A further construct consists of an AMP with gram-selectivity conjugated to the NBD fluorophore (AMP-2). These are combined with FCFM to allow distal alveolar imaging at micron resolution in anex vivoovine model of bacterial infection.ResultsAMP-1 demonstrates bacterial binding affinity in a concentration dependent manner and labels a diverse panel of bacteria, including a panel consisting of >70% of ventilator-associated pneumonia causing organisms and the pathogenic fungiAspergillus fumigatus.AMP-1 demonstrates significantly higher fluorescence over isomolar linear equivalents forE. coli, K. pneumoniae, P. aeruginosa, MSSA,A. baumanniiandS. pneumoniae(all p < 0.01), is selective for bacteria over mammalian cells and has improved chemical stability over the linear equivalent when incubated with bronchoaoveolar lavage from patients with acute respiratory distress syndrome. Furthermore, AMP-1 can labelE. coli, K. pneumoniae, P. aeruginosaand MSSAin situin anex vivoovine model when instilled endobronchially and imaged with FCFM (pin vitro and remains selective for gram-negative bacteria over mammalian cells. In theex-vivomodel AMP-2 selectively labels the gram-negative bacterial segments (P. aeruginosa, K. pneumoniaandE. coli) over the gram-positive (MSSA, MRSA andS. pneumoniae) or control pulmonary segments (all p < 0.05).ConclusionsA Smartprobe/FCFM strategy to immediately detect bacteria with gram selectivity in size relevant pre-clinical models is described, and are undergoing first-in-man translation.