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

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 replicationaltering 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. SignificanceThe 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.

Synthesis of novel pyrroles and fused pyrroles as antifungal and antibacterial agents

“…A question may arise about the effect that the compounds may have on the human DNA, but since the protein importance in the bacterial DNA replication is different from the eukaryotic counterparts, chromosome replication represents a promising therapeutic target 90,91 without such a fear on human safety 92 . There are many examples about the selectivity of the DNA acting antibacterial agents based on the structure difference of the targeted enzymes between bacteria and human, like quinolones which do not interfere with the human topoisomerase enzymes 93 , bacterial primase inhibitors 85 and Pol IIIC inhibitors 84 .…”