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.

Environmental and intracellular stresses can perturb protein homeostasis and trigger the formation and accumulation of protein aggregates. It has been recently suggested that the level of protein aggregates accumulated in bacteria correlates with the frequency of persister and viable but nonculturable cells that transiently survive treatment with multiple antibiotics. However, these findings have often been obtained employing fluorescent reporter strains. This enforced heterologous protein expression facilitates the visualization of protein aggregates but could also trigger the formation and accumulation of protein aggregates. Using microfluidics-based single-cell microscopy and a library of green fluorescent protein reporter strains, we show that heterologous protein expression favors the formation of protein aggregates. We found that persister and viable but nonculturable bacteria surviving treatment with antibiotics are more likely to contain protein aggregates and downregulate the expression of heterologous proteins. Our data also suggest that such aggregates are more basic with respect to the rest of the cell. These findings provide evidence for a strong link between heterologous protein expression, protein aggregation, intracellular pH, and phenotypic survival to antibiotics, suggesting that antibiotic treatments against persister and viable but nonculturable cells could be developed by modulating protein aggregation and pH regulation.

Persister and VBNC cells can phenotypically survive environmental stressors, such as antibiotic treatment, limitation of nutrients, and acid stress, and have been linked to chronic infections and antimicrobial resistance. It has recently been suggested that pH regulation might play a role in an organism’s phenotypic survival to antibiotics; however, this hypothesis remains to be tested.