IQUIBICEN   23947
INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CIENCIAS EXACTAS Y NATURALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Fluoromycobacteriophages for rapid TB diagnosis in sputum samples and phenotypic drug susceptibility testing?.
Autor/es:
PAYASLIAN FLORENCIA; LILIANA RONDON; ESTEFANIA URDANIZ; MARIANA PIURI
Reunión:
Congreso; XI SLAMTB 2018; 2018
Resumen:
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. Our aim is to develop a novel, rapid and sensitive assay to be used for in vitro activity testing of compounds. Fluorophages, mycobacteriophages carrying fluorescent genes, have become a useful tool to reveal the metabolic state of mycobacterial cells, and therefore their response to antibiotics. We optimized the conditions for automated fluorimetric detection with our fluorophage mCherrybombΦ. in a 96-well format as a good alternative for in vitro extracellular activity testing of compounds. A decrease in the fluorescent signal was observed over time for increasing concentrations of compounds with different targets in M.tb allowing MIC calculation. To validate the methodology for drug screening, we miniaturized the assay to 384-well plates and evaluated GSK?s TB set of compounds (n=232) comparing results between M.tb H37Rv o mc26230 and M. bovis BCG. We set up the best conditions for infection taking into account several parameters such as volume and UFC of inoculum, MOI, dispensation (manual vs. automated) and incubation time to achieve optimal fluorescence readout. Standard deviation and signal to background ratio (S:B) were not as good as expected, associated to a high background in the phage stock. Analysis were performed using software Spotfire (TIBCO) including a correlation with the results previously obtained with this set of compounds using REMA methodology and we could create an order for activity of compounds. As expected, after preincubation for 24 h of bacteria with drugs, compounds that showed best activity were those identified as inhibitors of essential membrane proteins, for example DprE, MmpL3, QcrB, and KasA. Using this methodology we were able to identify several interesting compounds with high potency and unknown mechanism of action that could be further explored. We are developing a new generation of luciferase reporter phages that would increase the difference between controls and increase the S:B ratio in order to get a rapid and robust assay for drug screening.