Temporal and intrinsic factors of rifampicin tolerance in mycobacteria

Abstract

Mycobacteria grow and divide asymmetrically, creating variability in growth pole age, growth properties, and antibiotic susceptibilities. Here, we investigate the importance of growth pole age and other growth properties in determining the spectrum of responses of Mycobacterium smegmatis to challenge with rifampicin. We used a combination of live-cell microscopy and modeling to prospectively identify subpopulations with altered rifampicin susceptibility. We found two subpopulations that had increased susceptibility. At the initiation of treatment, susceptible cells were either small and at early stages of the cell cycle, or large and in later stages of their cell cycle. In contrast to this temporal window of susceptibility, tolerance was associated with factors inherited at division: long birth length and mature growth poles. Thus, rifampicin response is complex and due to a combination of differences established from both asymmetric division and the timing of treatment relative to cell birth.antibiotic susceptibility | single cell | mathematical modeling | mycobacteria | cell biology

A Parallel Adder Coordinates Mycobacterial Cell-Cycle Progression and Cell-Size Homeostasis in the Context of Asymmetric Growth and Organization

“…In this and previous studies we used the SSB-GFP reporter to characterize the timing of M. smegmatis DNA replication (Figure 2E) [12, 17]. We found that the frequency and duration of E period correlates positively with birth length (Figure 2E) [7].…”

“…Using time-lapse microscopy, we observed one to two SSB-GFP foci during DNA replication (“C”) but none during pre- (“B”) and post- (“D”) replication periods (Figure 1H). We and others have observed M. smegmatis subpopulations that begin new rounds of replication after completing the first round but before division [10, 12, 17, 19]. We identified this additional replication period (“E”) in 54% of cells by observing SSB-foci near poles pre-division (Figures 1H, 1I&S1P).…”

“…M. smegmatis cells were filtered through a 10μm filter to remove clumps before being loaded into a custom polydimethylsiloxane (PDMS) microfluidic device, as in [32]. The viewing device contained a main microfluidic medium feeding channel, with a height of approximately 10–17μm, and viewing chambers with a diameter of 60 μm and a height of 0.8–0.9μm.…”

“…Cell poles and any visible foci were annotated each frame. In the majority of experiments, cell division was defined as “v-snapping” or “pinching”, as in [27, 32]. FM4-64 was also evaluated as a division determinant in Table S1.…”

“…If foci disappeared for a frame or two due to focus or cell overlap and then reappear, we assumed replication continued until the last foci disappears. SSB-GFP often exhibits diffuse background fluorescence when cells are not replicating DNA [2, 10, 12, 17, 18]. Cells within a microcolony can have varying levels of background SSB-GFP.…”