Bioluminescent reporter phage for the diagnostic detection of shigellosis

描述（由申请人提供）：志贺氏菌病是一个全球性的人类健康问题，也是一个值得关注的生物防御领域。这种由志贺氏菌属引起的疾病是发病和死亡的重要原因，全世界每年有1.64亿例病例和110万例死亡，其中最主要的是儿童。该疾病具有极强的传染性，仅需要10-100个细菌细胞即可开始感染。此外，多重耐药分离株的出现在全球范围内增加，例如流行性和大流行性志贺菌1型菌株。尽管有这种趋势，但用于确认疾病鉴定和确定抗生素敏感性的标准方法是传统的培养方法，然后是血清学和生物化学试验;这些试验需要24-48小时才能在实验室环境中完成。本研究的长期目标是开发一种快速、便携的诊断检测技术，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谱，这将使适当的治疗选择成为可能，从而改善患者预后。R21阶段将产生用于开发可检测志贺氏菌属的光标记志贺氏菌报告基因的原理验证结果。通过特定地赋予生物发光信号响应。目的1将鉴定和优先考虑具有种属特异性和广泛感染性的志贺菌。目的2将细菌luxAB报告基因整合到志贺菌噬菌体基因组的非必需位点，以产生luxAB标记的报告基因。在靶细菌存在的情况下，报告噬菌体与特定的细胞受体结合，注入它们的噬菌体DNA，并使用宿主的转录和翻译机制来产生荧光素酶。在底物添加后，可以容易地检测到随后的生物发光响应。在证明志贺氏菌报告基因具有必要的检测属性之后，R33赠款将把报告基因噬菌体技术发展成临床诊断， 志贺氏菌病。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物种;（i） 需要显著更少的细胞来获得阳性信号（更灵敏）;（iii）直接与临床样本一起起作用（不需要分离纯细菌培养物）;（iv）提供同时的抗生素敏感性数据（并帮助患者预后），以及（v）与简单的手持检测装置一起起作用（现场适用/电池操作系统）。生产报告噬菌体的成本和消耗成本是最小的。该测定不需要技术专业知识或处理。因此，我们认为该技术特别适合资源有限的环境，并且能够按照本RFA中规定的要求在非实验室环境中发挥作用。

DESCRIPTION (provided by applicant): 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 muti-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 serologicl 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 th 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; (i) require significantly fewer cells to achieve a positive signal (more sensitive); (iii) function dirctly 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.