Bioluminescent reporter phage for the diagnostic detection of shigellosis

志贺氏菌病是一个全球性的人类健康问题，也是一个令人担忧的生物防御领域。这种疾病由以下原因引起 志贺氏菌是导致疾病和死亡的重要原因，全世界有1.64亿个病例 每年有110万人死亡，其中最明显的是儿童。这种疾病传染性极强，需要 只有10-100个细菌细胞开始感染。此外，全球多药耐药的发生率呈上升趋势。 耐药菌株，如流行和流行的志贺氏菌1型菌株。尽管有这种趋势， 该病确认鉴定和抗生素检测的标准方法 敏感性，是传统的培养方法，然后是血清学和生化测试；这些测试 在实验室环境中需要24-48小时才能完成。 这项研究的长期目标是开发一种快速、便携的诊断分析技术，可以 检测志贺氏菌病的病原体。重要的是，诊断技术将能够同时 提供抗生素敏感性概况，这将使适当的治疗方案成为可能，从而导致 改善了患者的预后。R21阶段将为开发产生原则证明结果 能检测到志贺氏菌属的光标记志贺氏菌报告噬菌体。通过特别授予生物发光体 信号响应。目标1将识别和优先选择显示物种特异性和广泛性的志贺氏菌噬菌体 菌株传染性。目标2将把细菌LuxAB报告基因整合到优先考虑的非必需部位 志贺氏菌噬菌体基因组创造豪华AB标签报告噬菌体。在目标细菌存在的情况下， 记者噬菌体结合特定的细胞受体，注射其噬菌体DNA，并利用宿主S转录和 生产荧光素酶的翻译机器。在底物添加后，随后的生物发光 可以很容易地检测到响应。继志贺氏菌记者噬菌体具有 必要的检测属性，R33的拨款将把记者的噬菌体技术发展成临床 志贺氏菌病的诊断。 我们假设，生物发光噬菌体检测系统将能够：(I)快速(在几分钟内) 检测志贺氏菌的存在并区分不同物种；(Ii)需要显著较少的细胞来 实现正面信号(更灵敏)；(Iii)直接与临床标本起作用(不需要 分离纯细菌培养)；(Iv)提供并发的抗生素敏感性数据(并帮助患者 预后)，以及(V)使用简单的手持检测设备的功能(现场适当/电池供电 系统)。生产报告噬菌体的成本和消耗品成本都是最低的。化验结果并没有 需要专业的技术知识或加工。因此，我们认为这项技术特别适合于 资源有限的环境，能够在非实验室环境中按要求工作 在本RFA中阐述。

Shigellosis is a global human health problem and a biodefense area of concern. The disease, caused by Shigella species, is a significant cause of morbidity and mortality accounting for 164 million cases worldwide and 1.1 million deaths annually, most notably amongst children. The disease is extremely infectious, requiring only 10-100 bacterial cells to initiate infection. Moreover, there is a global rise in the occurrence of multi-drug resistant isolates, such as the epidemic and pandemic Shigella dysenteriae type 1 strain. Despite this trend, the standard methods for the confirmed identification of the disease and the determination of antibiotic susceptibility, are traditional culturing methods, followed by serological and biochemical tests; these assays require 24-48 h to complete in a lab environment. The long-term goal of this research is to develop a rapid, and portable diagnostic assay technology that can detect the causative agents of shigellosis. Importantly, the diagnostic technology will be able to simultaneously provide an antibiotic susceptibility profile, which will enable appropriate treatment options and thus lead to improved patient prognosis. The R21 phase will generate the proof-of-principle results for the development of ¿light-tagged¿ Shigella reporter phages that can detect Shigella spp. by specifically conferring a bioluminescent signal response. Aim 1 will identify and prioritize Shigella phages which display species specificity and broad strain infectivity. Aim 2 will integrate the bacterial luxAB reporter genes into non-essential sites of the prioritized Shigella phage genomes to create luxAB-tagged reporter phages. In the presence of target bacteria, the reporter phage bind to specific cell receptors, inject their phage DNA, and use the host¿s transcriptional and translational machinery to produce the luciferase enzyme. Upon substrate addition, the ensuing bioluminescent response can be readily detected. Following the demonstration that the Shigella reporter phages have the necessary detection attributes, the R33 grant will develop the reporter phage technology into a clinical diagnostic for shigellosis. We hypothesize that the ¿bioluminescent¿ phage detection system will be able to: (i) rapidly (within minutes) detect the presence of Shigella and differentiate between the species; (ii) require significantly fewer cells to achieve a positive signal (more sensitive); (iii) function directly with clinical specimens (does not require the isolation of pure bacterial cultures); (iv) provide concurrent antibiotic susceptibility data (and help patient prognosis), and (v) function with a simple handheld detection device (field appropriate/battery operated system). The cost of producing the reporter phage and the consumable costs are minimal. The assay does not require technical expertise or processing. Consequently, we believe the technology is particularly well suited to resource-limited settings and will be able to function in a non-laboratory environment as per the requirements set forth in this RFA.