Isolation and development of bioluminescent reporter phages for bacterial dysentery

Eur J Clin Microbiol Infect Dis. 2015 Feb;34(2):395-403. doi: 10.1007/s10096-014-2246-0. Epub 2014 Sep 25.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Ampicillin / pharmacology
  • Anti-Bacterial Agents / pharmacology
  • Bacteriophages / genetics
  • Bacteriophages / isolation & purification*
  • Ciprofloxacin / pharmacology
  • DNA, Viral / chemistry
  • DNA, Viral / genetics
  • Dysentery, Bacillary / diagnosis*
  • Dysentery, Bacillary / microbiology
  • Feces / microbiology
  • Genes, Reporter
  • Humans
  • Luciferases / analysis
  • Luciferases / genetics
  • Luminescent Measurements
  • Sequence Analysis, DNA
  • Shigella dysenteriae / isolation & purification*
  • Shigella flexneri / virology*
  • Wastewater / virology

Substances

  • Anti-Bacterial Agents
  • DNA, Viral
  • Waste Water
  • Ciprofloxacin
  • Ampicillin
  • Luciferases