Diagnostic Bioluminescent Phage for Detection of Yersinia pestis

Abstract

Yersinia pestis is the etiological agent of the plague. Because of the disease's inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak, whether it is natural or deliberate, be detected and diagnosed quickly. The objective of this research was to generate a recombinant luxAB ("light")-tagged reporter phage that can detect Y. pestis by rapidly and specifically conferring a bioluminescent signal response to these cells. The bacterial luxAB reporter genes were integrated into a noncoding region of the CDC plague-diagnostic phage A1122 by homologous recombination. The identity and fitness of the recombinant phage were assessed through PCR analysis and lysis assays and functionally verified by the ability to transduce a bioluminescent signal to recipient cells. The reporter phage conferred a bioluminescent phenotype to Y. pestis within 12 min of infection at 28°C. The signal response time and signal strength were dependent on the number of cells present. A positive signal was obtained from 10 2 cells within 60 min. A signal response was not detectable with Escherichia coli, although a weak signal (100-fold lower than that with Y. pestis) was obtained with 1 (of 10) Yersinia enterocolitica strains and 2 (of 10) Yersinia pseudotuberculosis strains at the restrictive temperature. Importantly, serum did not prevent the ability of the reporter phage to infect Y. pestis, nor did it significantly quench the resulting bioluminescent signal. Collectively, the results indicate that the reporter phage displays promise for the rapid and specific diagnostic detection of cultivated Y. pestis isolates or infected clinical specimens.

Development of an Engineered Bioluminescent Reporter Phage for Detection of Bacterial Blight of Crucifers

“…alisalensis PBSPCA1 phage. V. harveyi luxAB was used as the reporter of choice because (i) luxAB has been successfully used as a reporter for phage-mediated detection of Gram-positive and -negative pathogens (3,19,22,32,33), (ii) bioluminescent signals may be easily visualized by a photon detection device (e.g., luminometer), and (iii) minimal processing of the sample is required (the only requirement is the addition of the substrate n-decanal).…”

“…To reduce the time to detection and to enable detection in complex matrices such as food, environmental, or clinical specimens, reporter phages are being developed as biodetectors of pathogenic bacteria (34). Reporter phages for E. coli, Listeria monocytogenes, Y. pestis, B. anthracis, Salmonella spp., and M. tuberculosis are effective for the detection of bacteria in a range of complex matrices such as milk, ground beef, spinach, soft cheese, and human serum and sputum (2,3,22,23,27,29,32,33,36). Although phages had not been previously developed for the detection of plant-pathogenic bacteria, they have received EPA registration for use as biopesticides on tomatoes and peppers to control bacterial spot caused by Xanthomonas campestris pv.…”

Viable Staphylococcus aureus Quantitation using 15N Metabolically Labeled Bacteriophage Amplification Coupled with a Multiple Reaction Monitoring Proteomic Workflow

“…The use of phages for bacterial detection is well documented (37). Bacteriophages are specific to their target host, self-replicate, have extensive shelf lives, are inexpensive (38), and infect only metabolically active cells (39).…”

Engineering of Bacteriophages Y2:: dpoL1-C and Y2:: luxAB for Efficient Control and Rapid Detection of the Fire Blight Pathogen, Erwinia amylovora

“…Common reporters are luciferases from bacteria or insects, fluorescent proteins, glycosidases, and others (16). Reporter phages have been designed for specific detection of various Gram-negative and Gram-positive species, such as E. coli (17)(18)(19)(20), Salmonella (21,22), Yersinia pestis (23), Shigella spp. (24), Listeria monocytogenes (25,26), Bacillus anthracis (27,28), and Mycobacterium spp.…”