Actively growing cells are the predominant persisters in exponential phase of Escherichia coli

Abstract

AbstractBacterial persistence is a phenomenon in which a small fraction of isogenic bacterial cells survives a lethal dose of antibiotics. It is generally assumed that persistence is caused by growth-arrested dormant cells generated prior to drug exposure. However, evidence from direct observation is scarce due to extremely low frequencies of persisters, and is limited to high persistence mutants or to conditions that significantly increase persister frequencies. Here, utilizing a microfluidic device with a membrane-covered microchamber array, we visualize the responses of more than 106 individual cells of wildtype Escherichia coli to lethal doses of antibiotics, sampling cells from different growth phases and culture media. We show that preexisting dormant persisters constitute only minor fractions of persistent cell lineages in populations sampled from exponential phase, and that most persistent cell lineages grew actively before drug exposure. Actively growing persisters exhibit radical morphological changes in response to drug exposure, including L-form-like morphologies or filamentation depending on antibiotic type, and restore their rod-like shape after drug removal. Incubating cells under stationary phase conditions increases both the frequency and the probability of survival of dormant cells. While dormant cells in late stationary phase express a general stress response regulator, RpoS, at high levels, persistent cell lineages tended to show low to moderate RpoS expression among the dormant cells. These results demonstrate that heterogeneous survival pathways may coexist within bacterial populations to achieve persistence and that persistence does not necessarily require dormant cells.