Watching antibiotics in action: Exploiting time-lapse microfluidic microscopy as a tool for target-drug interaction studies in Mycobacterium

Abstract

Spreading resistance to antibiotics and the emergence of multidrug-resistant strains have become frequent in many bacterial species, including mycobacteria. The genus Mycobacterium encompasses both human and animal pathogens that cause severe diseases and have profound impacts on global health and the world economy. Here, we used a novel system of microfluidics, fluorescence microscopy and target-tagged fluorescent reporter strains of M. smegmatis to perform real-time monitoring of replisome and chromosome dynamics following the addition of replication-altering drugs (novobiocin, nalidixic acid and griselimycin) at the single-cell level. We found that novobiocin stalled replication forks and caused relaxation of the nucleoid, nalidixic acid triggered rapid replisome collapse and compaction of the nucleoid, and griselimycin caused replisome instability with subsequent over-initiation of chromosome replication and over-relaxation of the nucleoid. This work is an example of using a microscopy-based approach to evaluate the activity of potential replication inhibitors and provides mechanistic insights into their modes of action. Our system also enabled us to observe how the tested antibiotics affected the physiology of mycobacterial cells (i.e., growth, chromosome segregation, etc.). Because proteins involved in the DNA replication are well conserved among bacteria (including mycobacterial species), the properties of various replication inhibitors observed here in fast-growing M. smegmatis may be easily extrapolated to slow-growing pathogenic tubercle bacilli, such as M. tuberculosis.\n\nSignificanceThe growing problem of bacterial resistance to antibiotics and the emergence of new strains that are resistant to multiple drugs raise the need to explore new antibiotics and re-evaluate the existing options. Here, we present a system that allows the action of antibiotics to be monitored at the single-cell level. Such studies are important in the light of bacterial heterogeneity, which may be enhanced in unfavorable conditions, such as under antibiotic treatment. Moreover, our studies provide mechanistic insights into the action modes of the tested compounds. As combined therapies have recently gained increased interest, it is also notable that our described system may help researchers identify the best combination of antimicrobials for use against infections caused by a variety of bacteria.