Existence of log-phase Escherichia coli persisters and lasting memory of a starvation pulse.

The vast majority of a bacterial population is killed when treated with a lethal concentration of antibiotics. The time scale of this killing is often comparable with the bacterial generation time before the addition of antibiotics. Yet, a small subpopulation typically survives for an extended period. However, the long-term killing dynamics of bacterial cells has not been fully quantified even in well-controlled laboratory conditions. We constructed a week-long killing assay and followed the survival fraction of Escherichia coli K12 exposed to a high concentration of ciprofloxacin. We found that long-term survivors were formed during exponential growth, with some cells surviving at least 7 d. The long-term dynamics contained at least three time scales, which greatly enhances predictions of the population survival time compared with the biphasic extrapolation from the short-term behavior. Furthermore, we observed a long memory effect of a brief starvation pulse, which was dependent on the (p)ppGpp synthase relA Specifically, 1 h of carbon starvation before antibiotics exposure increased the surviving fraction by nearly 100-fold even after 4 d of ciprofloxacin treatment.

Birth and Resuscitation of (p)ppGpp Induced Antibiotic Tolerant Persister Cells.

Transient antibiotic treatment typically eradicates most sensitive bacteria except a few survivors called persisters. The second messenger (p)ppGpp plays a key role in persister formation in Escherichia coli populations but the underlying mechanisms have remained elusive. In this study we induced (p)ppGpp synthesis by modulating tRNA charging and then directly observed the stochastic appearance, antibiotic tolerance, and resuscitation of persister cells using live microscopy. Different physiological parameters of persister cells as well as their regularly growing ancestors and sisters were continuously monitored using fluorescent reporters. Our results confirmed previous findings that high (p)ppGpp levels are critical for persister formation, but the phenomenon remained strikingly stochastic without any correlation between (p)ppGpp levels and antibiotic tolerance on the single-cell level. We could not confirm previous notions that persisters exhibit markedly low concentrations of intracellular ATP or were linked to post-transcriptional effects of (p)ppGpp through the activation of small genetic elements known as toxin-antitoxin (TA) modules. Instead, we suggest that persister cell formation under regular conditions is driven by the transcriptional response to increased (p)ppGpp levels.

Gene Expression Changes with Minor Effects on the Population Average Have Major Effects on the Occurrence of Cells with Extreme Protein Concentrations.

The cell-to-cell heterogeneity in a bacterial population provides a rich response to environmental changes and robust survival of an isogenic population. Especially, the rare, extreme phenotypes can be important for survival under transient lethal conditions. We analyze the probability of having an extremely high or low protein level in a stochastic model of gene expression. The fraction of rare state cells defined as the cells in the tails of distributions is found to be highly sensitive to small changes of the mean protein level. The result highlights the importance of relatively weak changes to the mean for the occurrence of rare phenotypes.