Understanding the metabolic impact of a multidrug resistant plasmid, pLL35, on diverse Escherichia coli lineages.

Antibiotic resistant infections are predicted to claim 10 million lives annually by 2050.
Multi-drug resistant bacterial strains cause untreatable infections or necessitate lastline antibiotics. Conjugative plasmids carry resistance genes and facilitate the
evolution of multidrug resistance by horizontal transfer of genetic material. Plasmids
have complex interactions with their bacterial hosts that are not fully understood.
Chapter 2 describes an analysis pipeline for untargeted metabolomics that was
developed and published to make this complex methodology more accessible, then
used throughout the thesis.
This thesis explores the metabolic relationship between the multi-drug resistant
plasmid pLL35 in diverse Escherichia coli strains across key stages: plasmid
acquisition, subsequent coevolution and exposure to antibiotics.
Chapter 3: Upon acquisition of the plasmid, metabolomics reveals strain specific
alterations in functions such as ubiquinone biosynthesis, central energy production and
amino acid biosynthesis.
Chapter 4: 3 strains that had been experimentally evolved in previous work were
compared to their ancestors and displayed metabolic alterations associated with
adaptation to lab conditions; amino acid biosynthesis, glycolysis and pyrimidine
biosynthesis. The strains had specific metabolic alterations according to evolutionary
treatment (plasmid carrying with and without cefotaxime selection) with a singular
exception: the global regulator cyclic AMP, which was consistently upregulated in
plasmid carriers in response to antibiotic selection. Functions affected by coevolution
and selection included amino acid biosynthesis, central energy production and stress
responses.
Chapter 5: 4 plasmid carrying strains are exposed to kanamycin, cefotaxime and
ciprofloxacin. Common stress and resistance responses are observed, but responses
at a pathway and metabolite level are strain specific in extent and direction of change.
Pathways affected include amino acid and nucleotide metabolism, energy production,
biosynthesis of cell wall and biofilm components and redox stress management,
providing insights into bacterial stress responses and resistance mechanisms beyond
plasmid-encoded genes. Thus, Escherichia coli adapt to plasmid carriage and
antibiotic exposure by subtle alterations of its metabolism on a network wide scale.