Novel Microfluidic Technologies to Interrogate Bacterial Pathogens with Single-Cell Precision
Goode, O
Date: 13 March 2023
Thesis or dissertation
Publisher
University of Exeter
Degree Title
PhD in Biological Sciences
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 ...
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.
Doctoral Theses
Doctoral College
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