Stringent Response Triggered by Valine-Induced Amino Acid Starvation Does Not Increase Antibiotic Tolerance in Escherichia coli Cultures Grown at Low Cell Density

The stringent response is a survival mechanism in bacteria that is activated by various environmental stressors, including nutrient limitation such as amino acid starvation. The hallmarks of the stringent response include slowed growth and decreased synthesis of translational machinery (tRNA and rRNA) in favour of increased expression of stress response genes. In Escherichia coli, these include genes involved in combating oxidative stress, such as catalase. Treatment of E. coli K-12 with excess valine is a well-established method of inducing isoleucine starvation to trigger the stringent response and has been previously correlated with increased tolerance to beta-lactams and aminoglycosides. In this study, the effect of the stringent response on antibiotic tolerance to ampicillin, kanamycin and ciprofloxacin were investigated using E. coli K-12 wild type (WG1), relA mutant (AT-2) and relA/spoT double mutant (AB301). Induction of the stringent response was confirmed using a floating disc assay to measure catalase activity and a Bradford assay to measure total protein. Short term and long term antibiotic resistance were assessed through the generation of growth curves following incubation of replete and starved cultures with sub-minimum inhibitory concentrations of antibiotic and minimum inhibitory concentration (MIC) assays. The results showed that under starvation conditions, catalase production was higher in wild type compared to the mutant strains, indicating that the stringent response had been induced, while total protein results were highly variable. Despite the increase in catalase production under starvation, no differences in MIC or growth were observed across replete and amino acid starved cultures when challenged with any of the three antibiotics, indicating that valine-induced isoleucine starvation did not have an effect on ampicillin, kanamycin or ciprofloxacin tolerance.