Survival of Escherichia coli after high-antibiotic stress is dependent on both the pregrown physiological state and incubation conditions

INTRODUCTION: The survival of bacterial cells exposed to antibiotics depends on the mode of action, the antibiotics concentration, and the duration of treatment. However, it also depends on the physiological state of the cells and the environmental conditions. In addition, bacterial cultures contain sub-populations that can survive high antibiotic concentrations, so-called persisters. Research on persisters is challenging due to multiple mechanisms for their formation and low fractions, down to and below one millionth of the total cell population. Here, we present an improved version of the persister assay used to enumerate the amount of persisters in a cell population. METHODS: The persister assay with high antibiotic stress exposure was performed at both growth supporting and non-supporting conditions. Escherichia coli cells were pregrown to various growth stages in shake flasks and bench-top bioreactors. In addition, the physiological state of E. coli before antibiotic treatment was determined by quantitative mass spectrometry-based metabolite profiling. RESULTS: Survival of E. coli strongly depended on whether the persister assay medium supported growth or not. The results were also highly dependent on the type of antibiotic and pregrown physiological state of the cells. Therefore, applying the same conditions is critical for consistent and comparable results. No direct connection was observed between antibiotic efficacy to the metabolic state. This also includes the energetic state (i.e., the intracellular concentration of ATP and the adenylate energy charge), which has earlier been hypothesized to be decisive for persister formation. DISCUSSION: The study provides guides and suggestions for the design of future experimentation in the research fields of persisters and antibiotic tolerance.