Novel Microfluidic Technologies to Interrogate Bacterial Pathogens with Single-Cell Precision

Abstract

The aim of this thesis was to investigate persister and viable but non-culturable (VBNC) cell phenotypes using novel single-cell technologies. These phenotypes have been identified to play a role in biofilm survival and relapse of chronic infections. Recent reports linking persistence with the acceleration of antibiotic resistant evolution is extremely concerning. Researching these cells is difficult due to their low abundance and transient nature. Here, I utilise microfluidics and flow cytometry throughout my investigations to enable examination of these phenotypes without genetic or environmental manipulation. These novel technologies allow me to interrogate individual cells to better understand persister and VBNC cell formation, maintenance and response to stressors. I set out to investigate the intracellular pH of persister, VBNC and susceptible cells during ampicillin treatment within a clonal culture. I found persisters have a lower and narrower intracellular pH which they were able to maintain during ampicillin treatment in contrast to VBNC and susceptible cell populations. Next, I used mutant strains to determine the impact of tryptophan metabolism and indole signalling on the population structure and intracellular pH of these phenotypes. I combined this with transcriptomic analysis which identified expressional changes to aid in explaining the responses demonstrated. Protein aggregation has been identified as a potential factor involved in dormancy depth and tied to persister and VBNC cell phenotypes. I show that enforced ectopic GFP expression through the use of reporter E. coli strains, impacts the development of protein aggregation. Persister and VBNC cells in particular, which survive antibiotic treatment show a higher likelihood of protein aggregate formation. My data suggests that protein aggregates are more alkali compared to the rest of the cell. Persister and VBNC cells are not only identified during antibiotic stress but in response to other antimicrobials including disinfectants. I show the presence of persisters when treating Yersinia pseudotuberculosis with high concentrations of hydrogen peroxide. Taken together my results provide further evidence to understand persistence and VBNC cells.