| Summary: | To reveal rare phenotypes in bacterial populations, conventional microbiology tools should be advanced to generate rapid, quantitative, accurate, and high-throughput data. The main drawbacks of widely used traditional methods for antibiotic studies include low sampling rate and averaging data for population measurements. To overcome these limitations, microfluidic-microscopy systems have great promise to produce quantitative single-cell data with high sampling rates. Using <i>Mycobacterium smegmatis</i> cells, we applied both conventional assays and a microfluidic-microscopy method to reveal the antibiotic tolerance mechanisms of wild-type and <i>msm2570::Tn</i> mutant cells. Our results revealed that the enhanced antibiotic tolerance mechanism of the <i>msm2570::Tn</i> mutant was due to the low number of lysed cells during the antibiotic exposure compared to wild-type cells. This is the first study to characterize the antibiotic tolerance phenotype of the <i>msm2570::Tn</i> mutant, which has a transposon insertion in the <i>msm2570</i> gene—encoding a putative xanthine/uracil permease, which functions in the uptake of nitrogen compounds during nitrogen limitation. The experimental results indicate that the <i>msm2570::Tn</i> mutant can be further interrogated to reveal antibiotic killing mechanisms, in particular, antibiotics that target cell wall integrity.
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