Application of Fluorophages for TB Detection and Screening of Antitubercular Drugs

LILIANA RONDÓN; MARIANA PIURI; ESTEFANÍA URDÁNIZ; GRAHAM F HATFULL; MARCELO MARTÍ

Boston

Congreso; ASM MICROBE 2016; 2016

American Society for Microbiology

Tuberculosis (TB) is a major cause of human mortality. The emergence of resistant Mycobacterium tuberculosis (M.tb) strains has become a serious public health problem worldwide complicating treatment and control of the disease. Nowadays, there is a need for new and efficient anti-TB drugs. The bottleneck on the TB drug discovery pipeline still remains in the time-consuming activity testing of compounds. Our aim is to develop a novel, rapid and sensitive assay to be used for in vitro and in vivo drug susceptibility testing (DST) of compounds as part of the solution to the serious TB health problem.The development of fluorophages, mycobacteriophages carrying fluorescent genes, was described as a simple means of revealing the metabolic state of M. tb cells, and therefore their response to antibiotics. Recently, we have constructed an improved version of fluorophages with higher sensitivity and shorter time todetection of signal. Red bright M.tb cells were observed by fluorescent microscopy after only 5 hours of infection with the new mCherrybombΦ. We optimized the conditions for automated fluorimetric detection in a multiwell format as a good alternative for in vitro DST. Briefly, pure cultures of M.tb were infected at a MOI of 100 with mCherrybombΦ in the presence of the mostcommon anti-TB drugs. A decrease in the fluorescent signal was observed over time for increasing concentrations of compounds with different targets in the bacteria and MICs were succesfully determined.To test whether our methodology would also be useful for in vivo DST, we developed an infection assay in eukaryotic cells. We were able to detect intracellular M.tb by confocal microscopy after infection with mCherrybombΦ in THP-1 derived macrophages andpulmonary epithelial cell line A549. Basically, cells were incubated with bacteria and after extensive washing, mCherrybomb Φ was added overnight to reveal the presence of intracellular M.tb. Cells were stained with nuclear DAPI and inmunofluorescence was performed using a cytoplasmic antibody anti- LC3 autophagy marker. An automated infection assay in presence of different concentrations of drugs still must be evaluated. However, these promising results show that our phage-technology could be used for both in vitro and in vivo activity testing of compounds in a full-scale, fast and sensitive assay.