Cystic Fibrosis (CF) patients commonly are challenged by bacterial infections, including with non- tuberculosis Mycobacteria (NTM) such as Mycobacterium abscessus and Mycobacterium avium. These can present substantial clinical challenges because of their resistance to antibiotics and poor responsiveness to antibiotic therapy. Lung transplantation can substantially improve the severe pulmonary difficulties of CF, but immunosuppressive drugs supporting transplantation facilitate dissemination of NTM infection and high mortality rates. We recently reported the successful use of a bacteriophage cocktail to treat a 15-year old CF patient in London who had a bilateral lung transplant and suffered from a life-threatening M. abscessus infection. This study strongly suggests that if an effective cocktail of phages can be formulated for a specific patient infection, that a therapeutic intervention with phages can be very effective. However, the approach is not generally applicable to all NTM infections or even all M. abscessus infections, due to high strain variation in phage susceptibility profiles. That is, phages that are effective for one patient are not effective for other patients. The primary goal of this proposal is to develop a simple, quick, timely, and moderate-throughput assay for screening clinical isolates of NTB strains ? as well as M. tuberculosis strains ? for phage susceptibility. This assay also has the potential to monitor the emergence of phage resistance during the course of therapy. The assay is based on the simple concept of constructing a suite of candidate phages carrying a fluorescent reporter gene, such that fluorescence is emitted when the phages successfully infect a particular strain. Phage susceptibility can then be monitored in a microtiter plate format within a few hours, with simple discrimination between strains that are suspectable and those that are not. The challenge to constructing a suite of such phages has been addressed by developing a modified engineering strategy that simplifies the construction and broadens the types of phages that can be engineered. The reporter phage suite will be evaluated on a set of clinical isolates to determine their profiles of expression, sensitivity, and the feasibility of multiplexing phages with different `colored' reporters. Once validated, this reporter assay should facilitate the testing of large numbers of clinical NTM isolates to determine those for which phage intervention is a viable option. The ability to simply and quickly monitor the emergence of resistance during TB treatment will advance the prospects of TB clinical trials.
A current limitation to the therapeutic use of bacteriophages to treat mycobacterial infections is the great variability among clinical strains of M. abscessus and M. avium in their sensitivity to phages, and screening which strains are sensitive is both time-consuming and slow. A suite of fluorescent reporter phages will facility rapid strain screening in a high throughput format.
