Towards Second Generation of Fluoromycobacteriophages for Detection and Antibiotic Susceptibility Testing of Mycobacterium tuberculosis

MARIANA PIURI; ESTEFANIA URDANIZ; GRAHAM HATFULL

San Francisco

Congreso; 112th General Meeting American Society for Microbiology; 2012

ASM

Tuberculosis (TB) is a major cause of human mortality; almost two billion people are infected with the causative agent, Mycobacterium tuberculosis. There is a need for new diagnostic approaches that combine speed, sensitivity, specificity, biosafety, rapid and accurate determination of resistance to anti-tuberculosis drugs at a low cost to be applied in developing countries. We recently described the development of Fluoromycobacteriophages – reporter Mycobacteriophages containing a fluorescent reporter gene – that provide a simple means of revealing the metabolic state of M. tuberculosis cells, and their response to antibiotics.  Fluorescence can be detected easily by fluorescent microscopy or by flow cytometry and 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 is to develop the next generation of fluoromycobacteriophages that can be used for direct analysis of clinical samples. We propose to enhance the fluorescence signal to decrease the time-to-detection. We are constructing replication-proficient, lysis-defective phages. We have also created optimized versions of fluorescent genes with an enhanced mycobacterial expression., and 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.Finally, the construction of these optimized versions of Fluoromycobacteriophages will facilitate the testing of specific protocols for sputum processing to achieve efficient phage infection of mycobacterial cells directly in these samples. Together, these developments will result in a simple, rapid, and specific diagnostic test for tuberculosis.