Mycobacteriophages Engineered for TB Detection

MARIANA PIURI

Conferencia; 20th Biennial Evergreen International Phage Meeting; 2013

Mycobacteriophages are excellent candidates for the development of genetic and diagnostic tools since they efficiently and specifically infect and replicate in Mycobacteria. We have described the development of Fluoromycobacteriophages ? reporter phages containing a fluorescent reporter gene ? that provide a simple means of revealing the metabolic state of M. tuberculosis cells, and therefore their response to antibiotics. Fluorescence can be detected easily by fluorescent microscopy or by flow cytometry. The assay is responsive to antibiotics, and fluorescence is maintained for at least two weeks following fixation, increasing biosafety and facilitating storage or transportation of samples. Fluoromycobacteriophages have promising attributes in the research laboratory, and our goal was to develop the next generation of fluoromycobacteriophages that can be used for direct analysis of clinical samples. We have incorporated an optimized version of mCherry gene with an enhanced mycobacterial expression to increase the signal and shorten the time-to-detection.  We also developed a system for addition of affinity tags to phage particles to ensure efficient capture of mycobacterial cells, and thus optimize the sensitivity of detection. We used this improved version to test specific protocols for sputum processing to achieve efficient phage infection of mycobacterial cells directly in these samples. Together, these characteristics result in a simple, rapid, and specific diagnostic test for tuberculosis. Finally, since we were able to adapt the method to a multiwell format, we describe a further application for rapid whole cell screening of new antitubercular drugs.