Escherichia coli cells are primed for survival before lethal antibiotic stress

Abstract

Non-genetic factors can cause significant fluctuations in gene expression levels. Regardless of growing in a stable environment, this fluctuation leads to cell-to-cell variability in an isogenic population. This phenotypic heterogeneity allows a tiny subset of bacterial cells in a population, referred to as persister cells, to tolerate long-term lethal antibiotic effects by entering into a non-dividing, metabolically altered state. One fundamental question is whether this heterogeneous persister population is due to a pre-existing genetic mutation or a result of a transiently-primed reversible cell state. To explore this, we tested clonal populations starting from a single cell using a modified Luria–Delbrück fluctuation test. Through we kept the conditions the same, the diversity in persistence level among clones was relatively consistent: varying from ~ 60–100 and ~ 40–70 fold for ampicillin (Amp) and apramycin (Apr), respectively. Then we divided and diluted each clone to observe whether the same clone had comparable persister levels for more than one generation. Replicates had similar persister levels even when clones were divided, diluted by 1:20, and allowed to grow for ~ 5 generations. This result explicitly shows a cellular memory passed on for generations and eventually lost when cells are diluted to 1:100 and regrown (> 7 generations). Our result demonstrates 1) the existence of a small population prepared for stress ("primed cells") resulting in higher persister numbers, 2) the primed memory state is reproducible and transient, passed down for generations but eventually lost, and 3) a heterogeneous persister population is a result of a transiently-primed reversible cell state and not due to a pre-existing genetic mutation.

Probing transient memory of cellular states using single-cell lineages

“…However, we believe that the true potential of this generalized fluctuation test is in microbial systems, where (unlike mammalian cells) cells are readily grown from single cells with relatively fast doubling times, and there is a longstanding tradition of performing the Luria–Delbrück experiment. Working in this direction, we currently have several ongoing collaborations probing antibiotic tolerance across bacterial species ( Mycobacterium tuberculosis, Vibrio cholerae, Enterobacter cloacae , and Salmonella enterica ), and a recent study characterizes the transient priming of bacterial cells even before lethal antibiotic stress (Hossain et al, 2022 ).…”

An isogenicE. colipopulation gives rise to multiple persister phenotypes

“…Persister assay. The assay was done as previously described 11,24 . Briefly, overnight cultures were diluted 1/100 into fresh media and grown until they reached between OD 600 ~0.1-0.2.…”

“…Our recent work reveals that high persistence levels do not correlate with high short-term tolerance levels. Hence, it becomes imperative to use lethal antibiotic concentrations when investigating the transcriptional regulatory mechanisms of persisters 11 .…”

“…Our recent work reveals that high persistence levels do not correlate with high short-term tolerance levels. Hence, it becomes imperative to use lethal antibiotic concentrations when investigating the transcriptional regulatory mechanisms of persisters 11 . The genes related to antibiotic survival are heavily disputed because of conflicting results and different interpretations of data.…”

Reversibility of resistance in a fluctuation test experiment modifies the tail of the Luria-Delbrück distribution

“…However, several studies have shown that stochastic expression of specific proteins can lead to resistance to drug therapy arising transiently. In such case, cells switch to become resistant for several generations, but then switch back into a drug-sensitive state [2][3][4][5][6]. Several tools that exploit the colony-to-colony variation to estimate the rates of switching into and out of the resistant state have been recently developed [7,8].…”