Growth dependent single-cell response to DNA damaging antibiotics in Escherichia coli – RESISTANTE

Antimicrobial resistance is a growing global health issue with significant implications in terms of morbidity and mortality. Whilst antibiotic targets are usually very well defined, little is known about how antibiotic efficacy depends on the physiological state of bacteria. We aim to tackle this question by characterising mechanistically and at the single-cell level how the survival of Escherichia coli cells exposed to the clinically relevant antibiotic ciprofloxacin depends on their growth rate. This study will be performed on standard, walled, bacteria and on cells that have switched to a wall-deficient physiology, often referred to as L-form, and associated with chronic infection. Ciprofloxacin causes DNA double-strand breaks and is frequently used to treat infections. We have shown that fast-growing bacteria die more after exposure to ciprofloxacin than slow-growing ones. Moreover, the induction of DNA damage response (SOS response) varies depending on growth rate, which may impact survival. To uncover the underlying molecular mechanisms, we will (i) combine microfluidics and our established single molecule imaging protocol of the repair machinery to image DNA repair in real time and correlate with SOS induction in different nutrient conditions. DNA breaks will be induced using ciprofloxacin or a genetic system which allows us to create breaks in a controlled manner (ii) induce the switch to the slow-growing L-form physiology and characterise the response of L-form to ciprofloxacin at single cell level after designing and developing L-form adapted microfluidic devices. (iii) evaluate the effect of the strain’s genetic background by comparing the susceptibility to ciprofloxacin of a collection of UroPathogenic E. coli strains, which show significant differences in growth rate in identical nutrient conditions. We will also quantify their capacity to produce L-form and characterise their L-form response in the presence of ciprofloxacin. This project will provide a better understanding of the importance of clinically relevant physiological changes on the susceptibility of E. coli to DNA damage, with direct relevance to urinary tract infections.