Isolation of persisters enabled by ß-lactam-induced filamentation reveals their single-cell awakening characteristics

When exposed to lethal doses of antibiotics, bacterial populations are most often not completely eradicated. A small number of phenotypic variants, defined as ‘persisters’, are refractory to antibiotics and survive treatment. Despite their involvement in relapsing infections caused by major pathogens, processes determining phenotypic switches from and to the persister state largely remain elusive. This is mainly due to the low frequency of persisters in a population and the lack of reliable persistence markers, both hampering studies of persistence at the single-cell level. Problematically, existing methods to enrich for persisters result in samples with very low persister densities and/or a too high abundance of other cell types. Here we present a novel and highly effective persister isolation method involving cephalexin, an antibiotic that induces extensive filamentation of susceptible cells. We show that antibiotic-tolerant cells can easily be separated by size after a short cephalexin treatment, and that the isolated cells are genuine persisters. We used our isolation method to monitor persister outgrowth at the single-cell level in a microfluidic device, thereby conclusively demonstrating that awakening is a stochastic phenomenon. We anticipate that our novel approach can have far-reaching consequences in the persistence field, by allowing single-cell studies at a much higher throughput than previously reported.