Abstract
Antibiotic persistence occurs when a subpopulation of isogenic, antibiotic sensitive cells
survives prolonged exposure to a bactericidal concentration of an antibiotic. This
phenomenon has been shown to contribute to prolonged treatment duration, infection
recurrence, as well as accelerated development of genetic resistance. Several molecular
mechanisms have been demonstrated to influence formation of persister cells, however,
the direct casual links between these mechanisms and persister cell formation remain to
be established.
In this thesis we demonstrate that, contrary to previous reports, E. coli HipQ, a strain
created during an untargeted mutagenesis screen known to produce a high number of
antibiotic persisters, does not carry a mutation in the ydcI transcription factor.
Subsequently, we present evidence suggesting that ydcI does not regulate persistence
to β-lactams or fluoroquinolones. Furthermore, we show that the HipQ strain carries
multiple other non-synonymous substitutions, in contrast to previously reported two
single-nucleotide polymorphisms, including mutations in previously reported persistence
regulators, which are likely to contribute to its high-persistence phenotype.
Next we demonstrate, with the use of flow cytometry and an [NADH:NAD+ biosensor],
that ciprofloxacin exposure results in a high number of viable but non culturable cells
(VBNCs). This ciprofloxacin-tolerant VBNC subpopulation has lower intracellular
[NADH:NAD+], and therefore undergoes respiration at a lower rate, when compared to
the bulk population. Furthermore, we show that E. coli HipQ antibiotic-tolerant cells have
a significantly lower intracellular [NADH:NAD+] when compared to its parental strain,
which likely is a result of a mutation in the glycolysis pathway gap gene.
Finally, we assess antibiotic resistance, persistence and biofilm formation capabilities in
of a panel of urinary tract infection E. coli (UPEC) and demonstrate that in addition to
multidrug resistance, high-persistence to critically important antibiotics meropenem and
colistin is common amongst clinical isolates. We show that transient colistin resistance
develops rapidly when UPEC are incubated in in vivo mimicking conditions and that their
biofilm formation is dependent on attachment surface, with some isolates being able to
form biofilms on the surface of a uroepithelial cell monolayer, but not polystyrene.
‘Altogether, this thesis improves our understanding of molecular mechanisms regulating
the formation of E. coli antibiotic persisters, and further reaffirms the in-vivo relevancy of
persistence research.'