Second generation of Fluoromycobacteriophages for detection and antibiotic susceptibility testing of Mycobacterium tuberculosis

ESTEFANIA URDANIZ; GRAHAM F HATFULL; MARIANA PIURI

Mar del Plata

Congreso; VIII Congreso de Microbiología General; 2012

SAMIGE

Tuberculosis (TB) is a major cause of human mortality with 9 million new cases and nearly two million deaths annually; approximately two billion people are infected with the causative agent, Mycobacterium tuberculosis. In Argentina there are about 12,000 cases and one thousand deaths per year. While M. tuberculosis infections can be effectively resolved with a standard 6-9 month course of antibiotics with at least three drugs, the emergence of drug resistant strains severely complicates treatment. There is a need for new diagnostic approaches that combine speed (time-to-detection), sensitivity, specificity, biosafety, rapid and accurate determination of resistance to the commonly used anti-tuberculosis drugs at a low cost to be applied in developing countries where the incidence of TB is high. Mycobacteriophages are excellent candidates for the development of diagnostic tools since they efficiently and specifically infect and replicate in Mycobacteria. We recently 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 is to develop the next generation of fluoromycobacteriophages that can be used for direct analysis of clinical samples. In the first part of the project we propose to enhance the fluorescence signal to decrease the time-to-detection. To do so we are constructing replication-proficient, lysis-defective phages that also have the advantage of being used at any temperature, an attractive feature particularly useful in developing countries. We have created optimized versions of fluorescent genes with an enhanced mycobacterial expression that will be incorporated to the lysis-defective phages. 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.