Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria

Abstract

Phenotypic variability in populations of cells has been linked to evolutionary robustness to stressful conditions. A remarkable example of the importance of cell-to-cell variability is found in bacterial persistence, where subpopulations of dormant bacteria, termed persisters, were shown to be responsible for the persistence of the population to antibiotic treatments. Here, we use microfluidic devices to monitor the induction of fluorescent proteins under synthetic promoters and characterize the dormant state of single persister bacteria. Surprisingly, we observe that protein production does take place in supposedly dormant bacteria, over a narrow time window after the exit from stationary phase. Only thereafter does protein production stop, suggesting that differentiation into persisters fully develops over this time window and not during starvation, as previously believed. In effect, we observe that exposure of bacteria to antibiotics during this time window significantly reduces persistence. Our results point to new strategies to fight persistent bacterial infections. The quantitative measurement of single-cell induction presented in this study should shed light on the processes leading to the dormancy of subpopulations in different systems, such as in subpopulations of viable but nonculturable bacteria, or those of quiescent cancer cells.

Fig. 1.

Induction of single cells in a microfluidic device. (a) Current model for Type I persisters showing the formation of persisters at stationary phase. Cells arrest their growth at stationary phase. Upon transfer to fresh medium, normal cells grow and become sensitive, whereas persister cells remain in the growth arrested state acquired at stationary phase. (b) Expected fluorescence response to induction of normal cells (black curve) and persister cells (red curve), based on the current model for Type I persisters shown in a. (c) Schematic layout of the induction network: YFP is constitutively expressed from the chromosome; the addition of aTc releases the repression of the tet promoter and mCherry proteins are produced. (d–k) Induction experiment in a microfluidic device: at t = 0, hipA7 bacteria from O/N culture were introduced in the microfluidic device and subjected directly to fresh growth medium + inducer (aTc). (d) All bacteria constitutively express the YFP (here shown in green). (e) Induction of the mCherry protein is seen in the red fluorescence increase. (f–h) Addition of 100 μg/ml ampicillin kills growing bacteria. (i–k) Antibiotics washed and a single persister identified (white arrow). (Scale bar: 5 μm.) (l) mCherry fluorescence increase of typical normal (black squares) and persister (red squares) cells. Initial fluorescence increases as fast in persisters and normal cells, in contrast to the expected behavior shown in b. The delay in detection is mainly because of the relatively high background fluorescence (a.u., arbitrary units).

Fig. 2.

Automated quantitative analysis of single-cell induction curves. (a–e) High-magnification microscopy on agarose+aTc of MGYA7Z1/Ptet-mCherry. (a–c) Simultaneous pictures of the same field of view, where the growth of microcolonies initiated by two normally growing cells are monitored. (a) Phase contrast. (b) YFP (here shown in green). (c) mCherry-induced fluorescence. (d) Automatic detection and tracking of the fluorescent bacteria in pseudocolors. Each new cell is assigned an arbitrary color by the detection algorithm. Similar shades indicate bacteria that are closely related in their pedigree (see also Movie S1). (Scale bar: 5 μm.) (e) Output curves of the mCherry fluorescence increase from the automated procedure. Each curve represents one bacterium and its progeny (a.u., arbitrary units).

Fig. 3.

Comparison of fluorescence induction between normally growing and nongrowing cells. (a) Growth of microcolonies starting from single bacteria. Late-growing cells are marked in red. (Inset) Similar growth data for the induction of the Plac promoter with IPTG. (b) mCherry fluorescence increase for normal (black) and persister bacteria (red). (Inset) Similar fluorescence data for the induction of the Plac promoter with IPTG. (c) Histogram of the times to cross a fluorescence threshold of five times the background level for normally growing (black) and slow-growing cells (red). The two peaks overlap and have similar median times. Histograms normalized to the total number of cells in a single experiment (136). Five independent experiments were conducted, all leading to similar results. (a.u., arbitrary units).

Fig. 4.

No response to induction in persister cells after the onset of dormancy. At t = 0, MGYA7Z1/Ptet-mCherry bacteria from O/N culture were introduced in the microfluidic device and subjected first to fresh growth medium without inducer. The inducer (aTc) was added only after 2.5 h. (a) All bacteria constitutively express YFP (here shown in green). (b and c) Induction and production of the mCherry protein are seen in the red fluorescence increase and in normal cells only. (d) Growing bacteria die because of addition of 100 μg/ml ampicillin. (e–g) Removal of ampicillin: a persister (white arrow) reverts to normal growth and divides. (Scale bar: 5 μm.) (h) Analysis of mCherry fluorescence increase of typical normal (black squares) and persister cells (red squares). Persister bacteria do not respond to the induction signal when applied, but only when they switch to normally growing cells. (a.u.: arbitrary units).

Fig. 5.

Decreased persistence level at the exit from stationary phase. (a) An O/N culture of MGYA7Z1/Ptet-mCherry bacteria was diluted in fresh LB medium and its growth monitored by MPN (black). At each time point, an aliquot of the culture was subjected to ampicillin and the number of persisters counted by MPN (red). During the lag period, an increase in the number of persisters is observed. Error bars are calculated from eight MPN replicates. Similar results were obtained for the hipA7 strain without plasmid (data not shown). (b) New model for Type I persisters showing the different stages in the formation of persisters: Cells arrest their growth at stationary phase. Upon transfer to fresh medium, all cells become sensitive and respond to the induction signal. After a typical time of ≈ 1 h, persisters fully differentiate into the dormant state that protects them from ampicillin, whereas normal cells continue to grow. (c) Expected response to induction applied directly at the exit from stationary phase, for normal cells (black curve), and persister cells (red curve), based on the new model for Type I persisters shown in b. (a.u., arbitrary units).