Timelapse microscopy of bacteria exposed to INH

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Timelapse microscopy of bacteria exposed to INH. • Representative timelapse microscopy experiment in which M. smegmatis expressing GFP was cultured in a microfluidic device under a constant flow of 7H9 medium and imaged for 212 hours at 15 min intervals, beginning with a single progenitor cell at t = 0 hours. Images were recorded on fluorescence (green) and phase channels, merged, and stacked to generate the movie. Selected snapshots from this movie are shown in Fig. 1A. INH (50 µg/ml) was added to the flow medium at t = 12-156 hours and t = 188-212 hours. • • Ref.: Dynamic Persistence of Antibiotic-Stressed Mycobacteria • Science 4 January 2013: Vol. 339 no. 6115 pp. 91-95 DOI: 10.1126/science.1229858 • http://www.sciencemag.org/content/339... • • Abstract • Exposure of an isogenic bacterial population to a cidal antibiotic typically fails to eliminate a small fraction of refractory cells. Historically, fractional killing has been attributed to infrequently dividing or nondividing persisters. Using microfluidic cultures and time-lapse microscopy, we found that Mycobacterium smegmatis persists by dividing in the presence of the drug isoniazid (INH). Although persistence in these studies was characterized by stable numbers of cells, this apparent stability was actually a dynamic state of balanced division and death. Single cells expressed catalase-peroxidase (KatG), which activates INH, in stochastic pulses that were negatively correlated with cell survival. These behaviors may reflect epigenetic effects, because KatG pulsing and death were correlated between sibling cells. Selection of lineages characterized by infrequent KatG pulsing could allow nonresponsive adaptation during prolonged drug exposure.

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