Novel technologies to study single-cell response to environmental stimuli

Antibiotic tolerant phenotypes, such as persister and viable but non culturable cells (VBNC), are known to be present in isogenic bacterial populations. These phenotypes are now recognised as an important factor in the recalcitrance of infections and the development of antibiotic resistance; which itself is currently a major global health crisis. However, despite their clinical importance, we still know little about the mechanisms behind their formation and the relationship between the two phenotypes. Due to the relatively low abundance of the two phenotypes within the population and, in the case of VBNC cells, their ability to remain dormant for extended periods of time, high throughput single cell approaches currently provide the best opportunities for investigating them; in particular microfluidics has emerged as an exciting platform for investigating phenotypic heterogeneity at the single cell level due to the control it allows of the extracellular environment. Using antibiotic persistence as a proxy, we identify temporal windows in which a growing E. coli population exhibits significant changes in phenotypic heterogeneity and determine highly regulated genes and pathways at the population level. We then develop a high throughput microfluidic protocol, based on the pre-existing Mother Machine device, to investigate persister and VBNC cells before, during and after antibiotic exposure at the single cell level. We then developed the first fully automated image analysis pipeline that is capable of analysing Mother Machine images acquired in both bright field and phase contrast imaging modalities. The combination of our protocol and image analysis software allowed us to investigate the role of the previously identified genes in the formation of antibiotic persister and VBNC cells, where we identify potential biomarkers for these phenotypes before exposure to antibiotic. We then used the microfluidic set up to investigate the relationship between protein aggregation and antibiotic persister and VBNC cells. We find that protein aggregation can be correlated to the expression of exogenous proteins and that cells containing visible protein aggregates are, in turn, more likely to be persister or VBNC cells; providing further evidence that these phenotypes are not distinct and are instead part of one physiological continuum.