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. 2024 Sep 13;12(9):1888.
doi: 10.3390/microorganisms12091888.

Characterization of Escherichia coli Persisters from Biofilm Culture: Multiple Dormancy Levels and Multigenerational Memory in Formation

Hirona Ikeda  1 , Sumio Maeda  1
Affiliations

Characterization of Escherichia coli Persisters from Biofilm Culture: Multiple Dormancy Levels and Multigenerational Memory in Formation

Hirona Ikeda et al. Microorganisms. .

Abstract

Persister cells (PCs), a subpopulation occurring within normal cells, exhibit a transient tolerance to antibiotics because of their dormant state. PCs are categorized into two types: type I PCs, which emerge during the stationary phase, and type II PCs, which emerge during the logarithmic phase. Using the conventional colony-forming method, we previously demonstrated that type I PCs of Escherichia coli form more frequently in air-solid biofilm culture than in liquid culture. In the current study, we modified a cell filamentation method as a more efficient and rapid alternative for quantifying PCs. This modified method yielded results consistent with those of the conventional method with 103-104 times higher sensitivity and less detection time, within several hours, and further revealed the existence of multiple levels of type I PCs, including a substantial number of deeply dormant cells. This study also discovered a potential epigenetic memory mechanism, spanning several generations (four or six cell divisions), which influences type II PC formation based on prior biofilm experience in E. coli.

Keywords: Escherichia coli; dormancy; epigenetic memory; modified cell filamentation method; persister cells; viable but nonculturable cells.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Schema of the experimental flow of the modified filamentation method. Cep treatment of the cell population produces filamentous non-PCs in addition to unaltered PCs and dead cells. Subsequent cell staining with PI or CTC can detect live and dead cells, enabling the identification of PCs as PI-negative nonfilamentous cells or CTC-positive nonfilamentous cells. Details of the experimental conditions are described in Materials and Methods Section 2.4.
Figure 2
Figure 2
Micrographs of cells stained with PI (A,C) or CTC (E,G), as observed under a fluorescence microscope. The corresponding phase-contrast microscopy images are shown in (B,D,F,H). These images were obtained after applying the filamentation method to detect the PCs-I of BC-derived MG1655 cells. The filamentation method identified four types of cells: filamentous living cells (FL), filamentous dead cells (FD), nonfilamentous living cells (NL, representing PCs-I, which are circled in yellow, with solid lines indicating visible cells and dotted lines indicating nonvisible cells), and nonfilamentous dead cells (ND). (C,D,G,H) are magnified images focusing on “NL” in panels (A,B,E,F), respectively.
Figure 3
Figure 3
Comparison of PCs-I between cells derived from LC (white bars) and BC (gray bars) across three E. coli strains [MG1655 (A–C), W3110 (D–F), and BW25113 (G–I)]. The comparison was performed using three detection protocols: filamentation method-PI (A,D,G), filamentation method-CTC (B,E,H), and colony method (C,F,I). The PC-I incidence (%) data are presented as means and standard deviations (S.D.). Statistical significance was determined using a t-test (* p < 0.05; ** p < 0.01; *** p < 0.005, n = 7–30).
Figure 4
Figure 4
Comparison of PCs-I between the three detection protocols (PI: filamentation method-PI, CTC: filamentation method-CTC, and Colony: colony method) in cells derived from LC (white bars) and BC (gray bars) across three E. coli strains ((A) MG1655, (B) W3110, and (C) BW25113). The PC-I incidence (%) data are presented as means and S.D. Statistical significance was determined using one-way ANOVA followed by a t-test between each group (*** p < 0.005, n = 7–30, NS: not significant).
Figure 5
Figure 5
Resuscitation of deeply dormant PCs-I derived from BC of MG1655 by supplementation of nutrients [A: 4 mM sodium pyruvate, B: 16 mM methyl pyruvate, C: 0.5% (v/v) glycerol, and D: 0.1% (w/v) glucose]. The results are compared with controls [colony-forming PCs-I (Colony), PI-negative PCs-I (PI), and CTC-positive PCs-I (CTC)]. The data are presented as a box-and-whisker plot.
Figure 6
Figure 6
Schema of multiple dormancy levels of PCs-I revealed via the filamentation method. This illustrates a probable relationship of three groups of PCs [PI-negative PCs-I (PI), CTC-positive PCs-I (CTC), and colony-forming PCs-I (Colony)], all of which exist on a gradually dormant continuum.
Figure 7
Figure 7
Micrographs of cells stained with PI (A,C) or CTC (E,F), as observed under a fluorescence microscope. The corresponding phase-contrast microscopy images are shown in (B,D,F,H). These images were obtained after applying the filamentation method to detect the PCs-II of BC-derived MG1655 cells. The filamentation method identified four types of cells: filamentous living cells (FL), filamentous dead cells (FD), nonfilamentous living cells (NL, representing PCs-II, which are circled in yellow, with solid lines indicating visible cells and dotted lines indicating nonvisible cells), and nonfilamentous dead cells (ND). (C,D,G,H) are magnified images focusing on “NL” in panels (A,B,E,F), respectively.
Figure 8
Figure 8
Comparison of PCs-II between cells derived from LC (white bars) and BC (gray bars) of MG1655 after 4 or 6 cell divisions (left panels (A,C) and right panels (B,D), respectively). The comparison was performed using the filamentation method-PI (A,B) and the filamentation method-CTC (C,D). Time courses of absolute numbers of PI-negative PCs (E) or CTC-positive PCs (F) per culture of BC- and LC-derived cells (0 h—culture start point, 2–6 divisions—number of cell divisions in the log phase, and 24 h—culture endpoint reaching the stable stationary phase). The PC incidence (%) data are presented as means and standard deviations (S.D.). Statistical significance was determined using a t-test (* p < 0.05; *** p < 0.005, n = 8–15).
Figure 9
Figure 9
Effect of the first PC-II selection on the second PC-II formation ((A) schema of the experimental flow; (B) data graph) and the effect of extracellular materials from BC or LC on PC-II formation (C). In panel B, labels below each bar denote the experimental manipulations in each data set. For example, “LC” denotes LC followed by PC-II selection and detection via the filamentation method-PI; “1st B to 2nd L” denotes the first BC followed by the first PC-II selection and colonization and then the second LC followed by the second PC-II selection and detection using the filamentation method-PI. The other samples are denoted in the same manner. In panel C, labels below each bar denote the experimental manipulations in each data set. “LC” and “BC” denote PC-II formation in LC- and BC-derived cells with no supply, respectively. “LC + BC EM” denotes PC-II formation in LC-derived cells supplied with extracellular materials of BC, and “BC + LC EM” denotes PC-II formation in BC-derived cells supplied with extracellular materials of LC. In panels B and C, data regarding the PC-II incidence (%) are presented as means and S.D. Statistical significance was determined using one-way ANOVA followed by a t-test between each group (* p < 0.05; ** p < 0.01; *** p < 0.005, n = 7–9, NS: not significant).
Figure 10
Figure 10
Conceptual diagram of the potential presence of an epigenetic memory mechanism in PC-II formation. This illustrates how the influence of the prior BC and LC in the stationary phase on the later PC-II formation in the logarithmic phase appears to extend beyond multiple generations of cell division.
See this image and copyright information in PMC

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