Viable but Nonculturable and Persister Cells Coexist Stochastically and Are Induced by Human Serum

Abstract

Dormancy holds a vital role in the ecological dynamics of microorganisms. Specifically, entry into dormancy allows cells to withstand times of stress while maintaining the potential for reentry into an active existence. The viable but nonculturable (VBNC) state and antibiotic persistence are two well-recognized conditions of dormancy demonstrated to contribute to bacterial stress tolerance and, as a consequence, yield populations that are tolerant to high-dose antibiotics. Aside from this commonality, more evidence is being presented that indicates the relatedness of these two states. Here, we demonstrate that VBNC cells are present during persister isolation experiments, further indicating that these cells coexist and are induced by the same conditions. Interestingly, we reveal that VBNC cells can exist stochastically in unstressed growing cultures, a finding that is characteristic of persisters. Furthermore, human serum induces the formation of both VBNC cells and persisters, a finding not previously described for either dormancy state. Lastly, we describe the role of toxin-antitoxin systems (TAS) in the induction of the VBNC state and report that these TAS, which are classically implicated in persister cell formation, are also induced during incubation in human serum. This study provides evidence for the recently proposed "dormancy continuum hypothesis" and substantiates the physical and molecular relatedness of VBNC and persister cells in a standardized model organism. Notably, these results provide new evidence for the clinical significance of VBNC and persister cells.

FIG 1

Induction of the VBNC state from persister cells. Log-phase cells of V. vulnificus were treated with ampicillin to isolate persister cells, which were then washed and incubated at 4°C to induce the viable but nonculturable state. Once cells were determined to be nonculturable (third bar; arrow indicates that cells were below level of detection), cultures were incubated at 20°C for 24 h to allow for resuscitation. Error bars represent standard errors of three biological replicates. Different letters indicate statistically significant differences.

FIG 2

VBNC cells resuscitate after antibiotic treatment. Log-phase cells were treated with ampicillin to isolate persister cells, which were then washed and either incubated at 20°C for 24 h to resuscitate VBNC cells (third bar) or incubated at 4°C to induce the VBNC state in persister cells prior to resuscitation (fourth bar). Error bars represent standard errors of three biological replicates. Different letters indicate statistically significant differences.

FIG 3

VBNC cells are present stochastically and coexist with persisters. V. vulnificus (A) and E. coli (B) persister cells were isolated from log-phase cells and then washed and allowed to resuscitate for 24 h at 20°C. Culturability was assessed on HI agar (as log CFU/ml) while viability (number of cells staining green) was assessed using a Live/Dead viability staining kit. Error bars represent standard errors of three biological replicates. Asterisks represent statistically significant differences between corresponding values for live and culturable cells (P < 0.05, by one-way ANOVA).

FIG 4

Human serum induces persister and VBNC cells. (A) Cells were incubated in active and inactive human serum (HS) for 1 h, before supplementation with ampicillin for 4 h to isolate persisters. Serum and antibiotics were then removed, and cells were allowed to resuscitate for 24 h at 20°C. Error bars represent standard errors of three replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (individual unpaired t test). (B) Persister cells were isolated at 30°C and 37°C. Error bars represent standard errors of three replicates. *, P = 0.0385 (unpaired t test). Cont Pers, control persister; Resus, resuscitated.

FIG 5

The VBNC state induces expression of hipA toxins. Expression of hipA homologs hipA2 and hipA3 are elevated in VBNC cells relative to levels of culturable cells in log phase. Fold expression values were calculated relative to log phase expression and normalized using rplT as the reference gene. Error bars indicate standard deviations of three biological replicates and three technical replicates. Asterisks represent statistically significant differences assessed using a Kruskal-Wallis nonparametric ANOVA and Dunn's post hoc test. **, P < 0.01; ****, P < 0.0001.

FIG 6

The VBNC state induces expression of relE toxins. (A) Expression of relE homologs relE1-relE2, relE3, and relE5 are elevated in VBNC cells relative to levels in culturable cells. (B) Relative expression of relE5 (VV1_0110) is elevated in cells after 60 min of exposure to VBNC-inducing conditions. For both A and B, culturable cells are in log phase, and fold expression values were calculated relative to log-phase expression using rplT as the reference gene. Error bars indicate standard deviations of three biological replicates and three technical replicates. Asterisks represent statistically significant differences assessed using a Kruskal-Wallis nonparametric ANOVA and Dunn's post hoc test. *, P > 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 7

Human serum induces expression of TAS. Expression of relE5 and hipA2 was evaluated in cells incubated in active or inactive human serum for 4 h. Fold expression values were calculated relative to log-phase expression and normalized using gyrB as the reference gene. Error bars indicate standard deviations of three technical replicates. Asterisks represent statistically significant differences assessed using one-way ANOVA and Bonferroni's post hoc test. **, P < 0.01; ***, P > 0.001; ****, P < 0.0001.

FIG 8

The microbial dormancy continuum hypothesis. Environmental stress induces cellular processes that lead to the degradation of antitoxins, causing the liberation of their cognate toxins (triangles). This affects cellular metabolism and, consequently, growth. In the initial stages of dormancy, this produces antibiotic-tolerant nongrowing cells (persister cells) that can quickly resuscitate upon removal of the stress. Resuscitated or active cells have relatively low free-toxin levels and proliferate on medium. However, if cells are exposed to prolonged stress, more free toxins accumulate, the extent of dormancy increases, metabolic activity further decreases, and cells require more time to resuscitate, rendering cells viable but nonculturable (VBNC cells). When VBNC cells are allowed to resuscitate after removal of the inducing stress, their free-toxin levels slowly decrease while metabolic activity increases. At a particular free-toxin threshold, cells are defined as persisters because they are nongrowing but can quickly resuscitate into active cells and become culturable on medium. (Reprinted from reference with permission from Elsevier.)