Heterogeneous Distribution of Proton Motive Force in Nonheritable Antibiotic Resistance

Abstract

Bacterial infections that are difficult to eradicate are often treated by sequentially exposing the bacteria to different antibiotics. Although effective, this approach can give rise to epigenetic or other phenomena that may help some cells adapt to and tolerate the antibiotics. Characteristics of such adapted cells are dormancy and low energy levels, which promote survival without lending long-term genetic resistance against antibiotics. In this work, we quantified motility in cells of Escherichia coli that adapted and survived sequential exposure to lethal doses of antibiotics. In populations that adapted to transcriptional inhibition by rifampicin, we observed that ~1 of 3 cells continued swimming for several hours in the presence of lethal concentrations of ampicillin. As motility is powered by proton motive force (PMF), our results suggested that many adapted cells retained a high PMF. Single-cell growth assays revealed that the high-PMF cells resuscitated and divided upon the removal of ampicillin, just as the low-PMF cells did, a behavior reminiscent of persister cells. Our results are consistent with the notion that cells in a clonal population may employ multiple different mechanisms to adapt to antibiotic stresses. Variable PMF is likely a feature of a bet-hedging strategy: a fraction of the adapted cell population lies dormant while the other fraction retains high PMF to be able to swim out of the deleterious environment. IMPORTANCE Bacterial cells with low PMF may survive antibiotic stress due to dormancy, which favors nonheritable resistance without genetic mutations or acquisitions. On the other hand, cells with high PMF are less tolerant, as PMF helps in the uptake of certain antibiotics. Here, we quantified flagellar motility as an indirect measure of the PMF in cells of Escherichia coli that had adapted to ampicillin. Despite the disadvantage of maintaining a high PMF in the presence of antibiotics, we observed high PMF in ~30% of the cells, as evidenced by their ability to swim rapidly for several hours. These and other results were consistent with the idea that antibiotic tolerance can arise via different mechanisms in a clonal population.

Keywords: antibiotic resistance; beta-lactams; efflux pumps; flagellar motility; persistence.

FIG 1

Surviving proportion (left), motile proportion (middle), and mean speed of swimming cells (right) for the sequential exposure to rifampicin-ampicillin (R-A-exp), the single exposure to ampicillin (A-exp), and the single exposure to rifampicin (R-exp). No-exp refers to untreated wild-type cells. The means and standard errors for the CFU assays were calculated over three or more independent biological replicates (three technical replicates per biological sample). The differences in the mean CFU/mL across all the groups were significant. Motile proportions were determined from ~80 to 350 cells per replicate, and swimming speeds were determined from ~30 to 160 cells per replicate (over three biological replicates). The mean speeds were not significantly different across the different treatments, except for A-exp, where no motile cells were observed.

FIG 2

(A) Motile proportion and mean speed of swimming cells of the R-A-exp cells in the presence of ampicillin. The means and standard errors were calculated over three biological replicates. Motile proportions were determined from ~130 to 270 cells per replicate. Swimming speeds were determined from ~10 to 225 cells per replicate. (B) Cell length of R-A-exp, No-exp, and R-exp cells. The means and standard errors were calculated over three independent biological replicates with a total of 105 cells for each sample group. *, P < 0.05; **, P < 0.01.

FIG 3

(A) The mean fluorescence intensities are indicated for the R-A-exp and No-exp cells before (dark-gray bars) and after (hatched bars) the addition of CCCP. Emissions were averaged across three biological replicates and were collected from ~500 to 1,000 cells per replicate. (B) The relative membrane potential calculated from equation 1 and the relative mean swimming speeds are indicated by the black and white bars, respectively. The percentages of swimming cells are indicated by the light-gray bars. The means and standard errors were calculated over three biological replicates. **, P < 0.01. a.u., arbitrary units.

FIG 4

The rates of growth of different motile cells upon the removal of the antibiotic are plotted. The cell lengths (L) are normalized by their initial lengths (L₀) at the beginning of observation. Light curves indicate values for individual cells, and bold curves indicate mean values. Three types of motile cells are indicated: elongating-dividing cells (black curves, n = 11), elongating cells (blue curves, n = 3), and nonelongating-nondividing cells (red curves, n = 8).