φ²GFP10, a high-intensity fluorophage, enables detection and rapid drug susceptibility testing of Mycobacterium tuberculosis directly from sputum samples

The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific- fluorophage (φ²GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis in clinical sputum samples. Engineered features distinguishing φ²GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. φ²GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of φ²GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with φ²GFP10. Fluorophage φ²GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection and for rapid DST.