Heterogeneous Distribution of Proton Motive Force in Nonheritable Antibiotic Resistance

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.