Observation of non-dormant persister cells reveals diverse modes of survival in antibiotic persistence

Abstract

Bacterial 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.

Single-cell phenotypic characteristics of phenotypic resistance under recurring antibiotic exposure inEscherichia coli

“…In contrast to previously described characteristics of persiter cells, after release from antibiotics, we did not observe resumption of normal growth within 2-4 hours (3,4). Our findings hence support studies that explored persisters derived from exponentially growing cells that showed longer lag times to regain growth, note these cells had been growth arrest during drug exposure, or highlighting substantial variation in lag times after antibiotic exposure release (10,25,47). A lack of growth resumption, as we found, is also a characteristic of persister type II cells and VBNC cells, however VBNC are found in complete growth arrest and are characterized by a small cell size, and persister type II cells are not environmentally induced but predetermined (18,26), features not found in our study and other studies (10).…”

“…Our findings suggest additional diversity of modes of phenotypic resistance, given that we show previously undescribed combination of characteristics. Compared to previous persister studies that quantify fractions of <0.1% persisters (2–4,8,10,25), we reveal a substantial larger fraction (~25%) of cells that survived 1.5 hours of antibiotic exposure, independent of the level of antibiotics (Fig. 1a, see also findings at cell culture for <64 μg/ml; Fig.…”

“…Our phenotypic resistant cells cannot, a priori, be distinguished from susceptible cells by their reduced growth rate (26). Rare types of persister cells, that have previously described, did not arising from stationary phase cells and showed slow growth resumption post-exposure, these cells were almost growth arrested under antibiotic exposure (7,10), and we did not find such growth arrest for our cells.…”

“…Many of these single-cell studies use persister mutants, and start with growth-arrested, stationary phase cells, as these conditions increase the fraction of persister cells. Some recent studies suggest that persister cells arising of exponential growing populations are predominantly not growth arrested prior to antibiotic exposure (10). Despite evidence for diverse modes of phenotypic resistance, overall, these single-cell level studies highlight that that persister cells show limited growth when exposed to β-lactam or other antibiotics that inhibit cell wall formation or cell division (6,7,26), and that switching to persister stages can be irreversible or a reversible dynamic stochastic process (6,10).…”

“…Some recent studies suggest that persister cells arising of exponential growing populations are predominantly not growth arrested prior to antibiotic exposure (10). Despite evidence for diverse modes of phenotypic resistance, overall, these single-cell level studies highlight that that persister cells show limited growth when exposed to β-lactam or other antibiotics that inhibit cell wall formation or cell division (6,7,26), and that switching to persister stages can be irreversible or a reversible dynamic stochastic process (6,10). Earlier studies reported that the stochastic switch to the rare persister state occurred for growth-limited cells within exponentially growing E. coli populations, with ~80% of persisters arising from the non-growing sub-population prior to treatment, while the remaining persister cells originated from cells that were growing prior to treatment (11,27).…”