Age of Inoculum Strongly Influences Persister Frequency and Can Mask Effects of Mutations Implicated in Altered Persistence

Abstract

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...

Establishment of a Method To Rapidly Assay Bacterial Persister Metabolism

“…As evidence that the sedimented cells are persisters, Keren and colleagues performed LIVE/DEAD (SYTO/ PI) staining to show that all remaining cells stained as live cells (15). However, the claim that these sedimented cells are all persisters has come under scrutiny due to additional studies that have demonstrated that cell lysis by ␤-lactams leaves many more cells with intact membranes than there are persisters in the population (7,12,21). Here, we sought to determine if ␤-lactam lysis would be useful for isolation of wild-type E. coli persisters for metabolic measurements.…”

Targeting Persisters for Tuberculosis Control

“…Persister cells may have morphologies indistinguishable from those of susceptible cells or be distinct in some way, e.g., coccoid shape in old cultures and biofilms or cell wall-defective L-forms (85). The concept of persisters has evolved from the presumed homogeneous persister population (8) to much more heterogeneous and diverse populations (40,43,44,83) that are defined by specific conditions. Persisters are likely to comprise different subpopulations (40,44,83) and generally form a small and relatively predictable proportion of a culture.…”

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase

“…Clinically, persisters have been found to be important to the pathogenesis of infections, including persistent tuberculosis, reoccurring uropathogenic E. coli infections, and Pseudomonas aeruginosa infections in cystic fibrosis patients (10)(11)(12)(13)(14). The rate of persister formation is much higher in stationary-phase cells than in actively growing cells (10,(15)(16)(17)(18)(19). In addition to increasing rates of persister formation, stationary phase can also increase rates of resistance and tolerance to antibiotics (18,(20)(21)(22)(23).…”