Bioluminescent reporter phage for the diagnostic detection of shigellosis

用于志贺氏菌病诊断检测的生物发光报告噬菌体

• 批准号: 8300507
• 负责人: DAVID A SCHOFIELD
• 金额: $ 29.04万
• 依托单位: GUILD ASSOCIATES, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8300507
• 关键词: Accounting; Acute; Antibiotic susceptibility; Area; Bacteria; Bacteriophages; Binding; Biochemical; Biological Assay; Bioterrorism; Categories; Cause of Death; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Characteristics; Child; Clinical; Collection; Communicable Diseases; DNA; Data; Detection; Development; Devices; Diagnosis; Diagnostic; Disease; Drug resistance; Dysentery; Engineering; Environment; Enzymes; Epidemic; Freeze Drying; Generations; Genes; Genome; Goals; Grant; Growth; Hand; Health; Human; Infection; Lead; Light; Luciferases; Mediating; Methods; Morbidity - disease rate; Operating System; Patients; Phage Display; Phase; Phenotype; Process; Receptor Cell; Recombinants; Reporter; Reporter Genes; Research; Resources; Sampling; Serological; Shigella; Shigella Infections; Shigella boydii; Shigella dysenteriae; Shigella flexneri; Shigella sonnei; Signal Transduction; Site; Species Specificity; Specificity; Specimen; Staging; System; Technical Expertise; Technology; Testing; Translating; antimicrobial drug; biodefense; clinical Diagnosis; cost; enteric pathogen; fighting; improved; mortality; outcome forecast; pandemic disease; rapid detection; response; sample collection; tool; transmission process; trend

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. PUBLIC HEALTH RELEVANCE: Bacterial dysentery causes significant morbidity and mortality, especially in young children. This proposal will develop a diagnostic technology that can rapidly detect the bacterial agent that causes disease, and also provide information as to which antimicrobial drug is most effective in fighting the infection. Thus, this research has the potentil to significantly improve patient prognosis.

描述(申请人提供)：志贺氏菌病是一个全球性的人类健康问题，也是一个令人关注的生物防御领域。这种由志贺氏菌引起的疾病是发病率和死亡率的一个重要原因，每年全世界有1.64亿病例和110万人死亡，其中最明显的是儿童。这种疾病具有极强的传染性，只需要10-100个细菌细胞就能引发感染。此外，多药耐药菌株的出现在全球范围内呈上升趋势，例如流行和流行的1型志贺氏菌菌株。尽管有这一趋势，确认该病和确定抗生素敏感性的标准方法是传统的培养方法，然后是血清学和生化测试；这些测试需要24-48小时才能在实验室环境中完成。这项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，诊断技术将是 能够同时提供抗生素敏感性简档，这将使适当的治疗选择，从而导致改善患者的预后。R21阶段将为开发能够检测志贺氏菌的光标记志贺氏菌报告噬菌体产生原理证明结果。通过特别地授予生物发光信号响应。目的1对具有种属特异性和广谱菌株感染性的志贺氏菌噬菌体进行鉴定和筛选。目的2将细菌LuxAB报告基因整合到志贺氏菌噬菌体基因组的非必需部位，以创建LuxAB标记的报告噬菌体。在目标细菌存在的情况下，记者的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。在添加底物后，可以很容易地检测到随后的生物发光响应。在证明志贺氏菌报告噬菌体具有必要的检测属性后，R33赠款将把报告噬菌体技术发展为临床诊断 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) 实现阳性信号所需的细胞数量显著减少(更灵敏)；(Iii)直接与临床标本一起工作(不需要分离纯细菌培养)；(Iv)同时提供抗生素敏感性数据(并帮助患者预后)，以及(V)使用简单的手持式检测设备(现场适用/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。该检测不需要技术专业知识或处理。因此，我们认为这项技术特别适合于资源有限的环境，并将能够在非实验室环境中发挥作用，符合本RFA中提出的要求。 公共卫生相关性：细菌性痢疾导致严重的发病率和死亡率，尤其是在幼儿中。这项提议将开发一种诊断技术，可以快速检测导致疾病的细菌剂，并提供关于哪种抗菌药在对抗感染方面最有效的信息。因此，这项研究有可能显著改善患者的预后。