Single-cell physiological response of Escherichia coli to suppressive antibiotic combinations

Abstract

In the natural environment, bacteria are exposed to varying levels of nutrient availability which significantly affect cellular physiology and the efficacy of antibiotics. Changes in nutrient availability and growth rate also impose physiological constraints on the cell’s capacity to respond to stress, such as DNA damage, and can have important consequences on treatment outcomes, but this effect is under-appreciated. Bacteria deal with DNA damage by inducing the SOS response which plays an important role in survival under DNA stress and contributes to antibiotic resistance. In clinical practice, antibiotics are increasingly used in combination to improve efficacy against infectious diseases and to limit the emergence of resistance. For some antibiotic combinations, bacterial inhibition decreases when one antibiotic is added to another. This is called a suppressive drug interaction and is common between DNA synthesis inhibitors and translation inhibitors. Despite having a lower efficacy, suppressive drug combinations have potential in treating bacterial infections because they select against antibiotic resistance. However, the relationship between bacterial physiology and susceptibility to suppressive antibiotic combinations is not yet clearly understood.

Previous studies on SOS response induction and suppressive drug combinations rely on bulk-level experiments which lack insight into the underlying population dynamics and the inherent heterogeneity of single-cell growth and gene expression. Single-cell approaches are key in informing a mechanistic understanding of the cellular response to DNA damage and antibiotic combinations. In this thesis, we used a single-cell microfluidic device called the ‘Mother Machine’ and quantitative time-lapse microscopy to investigate growth-dependence in SOS response induction in Escherichia coli. We directly measured the rate of SOS induction in a strain experiencing sublethal, replication-dependent DNA damage and found that it was higher in fast-growth conditions. However, using a population dynamics model, we revealed that the proportion of high-SOS cells in a growing culture is not only a function of the rate of SOS induction but also depends upon competition with low-SOS, fast-dividing cells. Next, we investigated the physiological mechanisms that give rise to the suppressive interaction between ciprofloxacin (a DNA synthesis inhibitor) and tetracycline (a translation inhibitor). Our results show that an improvement in cell survival, rather than increased growth and division, is the dominant mechanism leading to suppression between ciprofloxacin and tetracycline. Furthermore, the suppressive effect was amplified in fast-growth conditions which could have important implications on treatments in fluctuating nutrient environments. Altogether, our results highlight the utility of single-cell time-lapse methods in revealing population dynamics and single-cell heterogeneity that would otherwise be masked in bulk-level methods. In furthering our understanding of bacterial responses to antibiotics and their combinations, we aim to contribute to the development of more effective treatment strategies.