Schofield D A, Wray D J, Molineux I J
Guild BioSciences, 1313B Ashley River Road, Charleston, SC, 29407, USA,
Eur J Clin Microbiol Infect Dis. 2015 Feb;34(2):395-403. doi: 10.1007/s10096-014-2246-0. Epub 2014 Sep 25.
Shigellosis is a significant cause of morbidity and mortality worldwide, most notably amongst children. Moreover, there is a global increase in the occurrence of multidrug-resistant isolates, including the epidemic and pandemic Shigella dysenteriae type 1 strain. We developed a bioluminescent reporter phage assay to facilitate detection and simultaneously determine antibiotic susceptibility. A Shigella flexneri phage (Shfl25875) was isolated from environmental wastewater and characterized by DNA sequencing. Shfl25875 is T4-like, harbors a 169,062-bp genome, and grows on most (28/29) S. flexneri strains and all 12 S. dysenteriae type 1 strains tested. The genes encoding bacterial luciferase were integrated into the Shfl25875 genome to create a "light-tagged" phage capable of transducing a bioluminescent phenotype to infected cells. Shfl25875::luxAB rapidly detects cultured isolates with high sensitivity. Specificity experiments indicate that the reporter does not respond to Shigella boydii, non-type 1 S. dysenteriae strains, and most non-Shigella Enterobacteriaceae. Shfl25875::luxAB generates ampicillin and ciprofloxacin susceptibility profiles that are similar to the standard Clinical and Laboratory Standards Institute (CLSI) growth microdilution method, but in a significantly shorter time. In addition, the reporter phage detects Shigella in mock-infected stool. This new reporter phage shows promise as a tool for the detection of cultured isolates or complex clinical samples.
志贺氏菌病是全球发病和死亡的重要原因，在儿童中尤为明显。此外，包括流行和大流行的痢疾志贺氏菌1型菌株在内的多重耐药菌株的全球发生率正在上升。我们开发了一种生物发光报告噬菌体检测方法，以促进检测并同时确定抗生素敏感性。从环境废水中分离出一株弗氏志贺氏菌噬菌体（Shfl25875），并通过DNA测序对其进行了表征。Shfl25875属于T4样噬菌体，基因组大小为169,062 bp，能在大多数（28/29）测试的弗氏志贺氏菌菌株和所有12株测试的痢疾志贺氏菌1型菌株上生长。将编码细菌荧光素酶的基因整合到Shfl25875基因组中，以创建一种“光标记”噬菌体，能够将生物发光表型传递给受感染的细胞。Shfl25875::luxAB能快速、高灵敏度地检测培养的分离株。特异性实验表明，该报告噬菌体对鲍氏志贺氏菌、非1型痢疾志贺氏菌菌株以及大多数非志贺氏菌属肠杆菌科细菌无反应。Shfl25875::luxAB生成的氨苄西林和环丙沙星敏感性谱与标准的临床和实验室标准协会（CLSI）生长微量稀释法相似，但所需时间明显更短。此外，报告噬菌体可在模拟感染的粪便中检测到志贺氏菌。这种新型报告噬菌体有望成为检测培养分离株或复杂临床样本的工具。
