?2GFP10: A high-intensity fluorophage enables detection and rapid drug susceptibility testing of Mycobacterium tuberculosis directly from sputum samples

Paras Jain, Travis E. Hartman, Nell Eisenberg, Max R. O'Donnel, Jordan Kriakov, Karnishree Govender, Mantha Makume, David S. Thaler, Graham F. Hatfull, A. Willem Sturm, Michelle H. Larsen, Preshnie Moodley and William R. Jacobs Jr.

The diagnosis of active tuberculosis (TB) and rapid drug susceptibility testing (DST) at point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterial-specific-fluorophage (φ2GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis (Mtb) 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. φ2GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampicin and kanamycin from sputum samples yielded results after 12h 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 as a rapid DST.