Fluoromycobacteriophages for Rapid, Specific, and Sensitive Antibiotic Susceptibility Testing of Mycobacterium Tuberculosis

PLoS One. 2009;4(3):e4870. doi: 10.1371/journal.pone.0004870. Epub 2009 Mar 20.

Abstract

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively-drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology*
  • Drug Resistance, Multiple, Bacterial*
  • Flow Cytometry / methods
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Humans
  • Microbial Sensitivity Tests / methods*
  • Mycobacteriophages / genetics
  • Mycobacteriophages / metabolism*
  • Mycobacterium smegmatis / metabolism
  • Mycobacterium tuberculosis / drug effects*
  • Sensitivity and Specificity

Substances

  • Anti-Bacterial Agents
  • Green Fluorescent Proteins