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。这种由志贺氏菌引起的疾病是发病率和死亡率的一个重要原因，全世界每年有1.64亿例病例和110万例死亡，其中儿童最为明显。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，全球出现耐多药分离株的情况有所增加，例如流行和大流行的志贺氏痢疾菌1型菌株。尽管有这种趋势，但确定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，其次是血清学和生化试验；这些检测需要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能够检测志贺氏菌病病原体的快速、便携式诊断检测技术。重要的是，诊断技术将

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.