title: Isolation and development of bioluminescent reporter phages for bacterial dysentery.
abstract: 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.
authors.affiliation: Guild BioSciences, 1313B Ashley River Road, Charleston, SC, 29407, USA, dschofield@guildbiosciences.com.
authors.forename: D A
authors.initials: DA
authors.lastname: Schofield
authors.forename: D J
authors.initials: DJ
authors.lastname: Wray
authors.forename: I J
authors.initials: IJ
authors.lastname: Molineux
mesh_terms: D000667:Ampicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001435:Bacteriophages / Q000235:genetics / Q000302:isolation & purification*; D002939:Ciprofloxacin / Q000494:pharmacology; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004405:Dysentery, Bacillary / Q000175:diagnosis* / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008163:Luminescent Measurements; D017422:Sequence Analysis, DNA; D012762:Shigella dysenteriae / Q000302:isolation & purification*; D012763:Shigella flexneri / Q000821:virology*; D062065:Wastewater / Q000821:virology
chemical_list: D000667:Ampicillin; D000900:Anti-Bacterial Agents; D002939:Ciprofloxacin; D004279:DNA, Viral; D008156:Luciferases; D062065:Wastewater