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Abstract Drug-recalcitrant infections are a leading global-health concern. Bacterial cells benefit from phenotypic variation, which can suggest effective antimicrobial strategies. However, probing phenotypic variation entails spatiotemporal analysis of individual cells that is technically challenging, and hard to integrate into drug discovery. In this work, we develop a multi-condition microfluidic platform suitable for imaging two-dimensional growth of bacterial cells during transitions between separate environmental conditions. With this platform, we implement a dynamic single-cell screening for pheno-tuning compounds, which induce a phenotypic change and decrease cell-to-cell variation, aiming to undermine the entire bacterial population and make it more vulnerable to other drugs. We apply this strategy to mycobacteria, as tuberculosis poses a major public-health threat. Our lead compound impairs Mycobacterium tuberculosis via a peculiar mode of action and enhances other anti-tubercular drugs. This work proves that harnessing phenotypic variation represents a successful approach to tackle pathogens that are increasingly difficult to treat.

Abstract Preclinical analysis of drug efficacy is critical for drug development. However, conventional bulk-cell assays statically assess the mean population behavior, lacking resolution on drug-escaping cells. Inaccurate estimation of efficacy can lead to overestimation of compounds, whose efficacy will not be confirmed in the clinic, or lead to rejection of valuable candidates. Time-lapse microfluidic microscopy is a powerful approach to characterize drugs at high spatiotemporal resolution, but hard to apply on a large scale. Here we report the development of a microfluidic platform based on a pneumatic operating principle, which is scalable and compatible with long-term live-cell imaging and with simultaneous analysis of different drug concentrations. We tested the platform with mycobacterial cells, including the tubercular pathogen, providing the first proof of concept of a single-cell dose–response assay. This dynamic in-vitro model will prove useful to probe the fate of drug-stressed cells, providing improved predictions of drug efficacy in the clinic.

Summary: RNA turnover is a primary source of gene expression variation, in turn promoting cellular adaptation. Mycobacteria leverage reversible mRNA stabilization to endure hostile conditions. Although RNase E is essential for RNA turnover in several species, its role in mycobacterial single-cell physiology and functional phenotypic diversification remains unexplored. Here, by integrating live-single-cell and quantitative-mass-spectrometry approaches, we show that RNase E forms dynamic foci, which are associated with cellular homeostasis and fate, and we discover a versatile molecular interactome. We show a likely interaction between RNase E and the nucleoid-associated protein HupB, which is particularly pronounced during drug treatment and infection, where phenotypic diversity increases. Disruption of RNase E expression affects HupB levels, impairing Mycobacterium tuberculosis growth homeostasis during treatment, intracellular replication, and host spread. Our work lays the foundation for targeting the RNase E and its partner HupB, aiming to undermine M. tuberculosis cellular balance, diversification capacity, and persistence.

There is an urgent need for new antimicrobials to treat the opportunistic Gram-negative Burkholderia cenocepacia, which represents a problematic challenge for cystic fibrosis patients. Recently, a benzothiadiazole derivative, C109, was shown to be effective against the infections caused by B. cenocepacia and other Gram-negative and-positive bacteria. C109 has a promising cellular target, the cell division protein FtsZ, and a recently developed PEGylated formulation make it an attractive molecule to counteract Burkholderia infections. However, the ability of efflux pumps to extrude it out of the cell represents a limitation for its use. Here, more than 50 derivatives of C109 were synthesized and tested against Gram-negative species and the Gram-positive Staphylococcus aureus. In addition, their activity was evaluated on the purified FtsZ protein. The chemical, metabolic and cellular stability of C109 has been assayed using different biological systems, including quantitative single-cell imaging. However, no further improvement on C109 was achieved, and the role of efflux in resistance was further confirmed. Also, a novel nitroreductase that can inactivate the compound was characterized, but it does not appear to play a role in natural resistance. All these data allowed a deep characterization of the compound, which will contribute to a further improvement of its properties.