The majority of cells transferred from stationary-phase culture into fresh medium resume growth quickly, while a few remain in a nongrowing state for longer. These temporarily nonproliferating bacteria are tolerant of several bactericidal antibiotics and constitute a main source of persisters. Several genes have been shown to influence the frequency of persisters in Escherichia coli, although the exact mechanism underlying persister formation is unknown. This study demonstrates that the frequency of persisters is highly dependent on the age of the inoculum and the medium in which it has been grown. The hipA7 mutant had 1,000 times more persisters than the wild type when inocula were sampled from younger stationary-phase cultures. When started after a long stationary phase, the two displayed equal and elevated persister frequencies. The lower persister frequencies of glpD, dnaJ, and surA knockout strains were increased to the level of the wild type when inocula aged. The mqsR and phoU deletions showed decreased persister levels only when the inocula were from aged cultures, while sucB and ygfA deletions had decreased persister levels irrespective of the age of the inocula. A dependency on culture conditions underlines the notion that during screening for mutants with altered persister frequencies, the exact experimental details are of great importance. Unlike ampicillin and norfloxacin, which always leave a fraction of bacteria alive, amikacin killed all cells in the growth resumption experiment. It was concluded that the frequency of persisters depends on the conditions of inoculum cultivation, particularly its age, and the choice of antibiotic.Genetically homogeneous bacterial cultures can give rise to subpopulations with different physiological properties (38). This kind of heterogeneity can be demonstrated in terms of growth resumption when stationary-phase bacteria are diluted in fresh medium: some cells start growth immediately, some later, and some do not recover during the period of observation (2, 15, 33). Variation in recovery could reflect the random nature of cell damage (8). Alternatively, as rapidly recovering bacteria are more vulnerable to harmful environmental conditions than dormant ones, the wide range of growth resumption times could represent an ecological strategy (11,21,39). For example, the majority of bactericidal antibiotics kill growing bacteria, and nongrowing cells survive (41). Furthermore, antibiotic-sensitive growing cultures cannot be sterilized by bactericidal drugs, suggesting the existence of a small subpopulation of nongrowing bacteria (4). These bacteria, called persisters, can survive antibiotic treatment and resume growth after removal of the drug (23).Several mutations lead to increased or decreased persister levels (9,10,13,19,24,25,27,40). A classical mutant with increased persister frequency is the hipA7 strain (27), which carries mutations in the gene coding for the toxin in the HipAB toxin-antitoxin pair (21). This mutation decreases the affinity of the toxin for anti...

Background: A fundamental characteristic of cells is the ability to divide. To date, most parameters of bacterial cultures, including cell division, have been measured as cell population averages, assuming that all bacteria divide at a uniform rate.

A genetically homogenous bacterial population may contain physiologically distinct subpopulations. In one such case, a minor part of an otherwise antibiotic-sensitive bacterial population maintains a nondividing state even in a growth-supporting environment and is therefore not killed by bactericidal antibiotics. This phenomenon, called persistence, can lead to failure of antibiotic treatment. We followed the development of sensitivity to killing by ampicillin and norfloxacin when Escherichia coli cells were transferred from a stationary-phase culture into fresh growth medium. In parallel, we monitored growth resumption by individual bacteria. We found that bacteria in a population resumed growth and became sensitive to antibiotics at different times after transfer to fresh medium. Moreover, both growing and dormant bacteria coexisted in the same culture for many hours. The kinetics of awakening was strongly influenced by growth conditions: inocula taken from the same stationary-phase culture led to very different persister frequencies when they were transferred into different fresh media. Bactericidal antibiotics kill cells that have woken up, but the later-awakening subpopulation is tolerant to them and can be identified as persisters when the antibiotic is removed. Our observations demonstrate that persister count is a dynamic measure and that the persister frequency of a particular culture is not a fixed value.

Frequent changes in nutrient availability often result in repeated cycles of bacterial growth and dormancy. The timing of growth resumption can differ among isogenic cells and delayed growth resumption can lead to antibiotic tolerant persisters. Here we describe a correlation between the timing of entry into stationary phase and resuming growth in the next period of cell proliferation. E. coli cells can follow a last in first out rule: the last ones to shut down their metabolism in the beginning of stationary phase are the first to recover in response to nutrients. This memory effect can last for several days in stationary phase and is not influenced by environmental changes. We observe that the speed and heterogeneity of growth resumption depends on the carbon source. A good carbon source (glucose) can promote rapid growth resumption even at low concentrations, and is seen to act more like a signal than a growth substrate. Heterogeneous growth resumption can protect the population from adverse effect of stress, investigated here using heat-shock, because the stress-resilient dormant cells are always present.