Cullen, Lucky Bonnie Lucia (2019) The Antibiotic Resistance Growth Plate (ARGP) as an experimental evolution tool to explore the phenotypic and genotypic mutational pathways underlying the emergence of antimicrobial resistance in 'Escherichia coli'. (PhD thesis), Kingston University, .
Abstract
The increasing threat of an antimicrobial resistance crisis is a significant global concern. Antimicrobial treatment failures are worsened by the rapid evolution of resistance amongst bacterial pathogens. Therefore, in addition to developing novel antimicrobial agents, there is growing interest in exploring the underlying genotypic factors of resistance evolution. Traditionally, such studies have focused heavily on well-established mechanisms of acquired resistance involving horizontal gene transfer, yet the evolution of resistance through the acquisitions of mutations is yet to be fully elucidated. Adaptive laboratory evolution studies have provided insights into the genetic basis of adaptation through the direct observation of the evolutionary process. Experimental evolution has advanced from serial passage in well mixed systems to the incorporation of spatiotemporal antibiotic concentration gradients. During this study the Antibiotic Resistance Growth Plate (ARGP) was developed as a simple experimental tool to explore the ability of bacteria to evolve resistance across an antibiotic landscape. The device (90mm × 15mm) facilitates the direct observation of the evolutionary trajectories of mutational lineages within a circular format supporting the radial growth and the exploration of phenotypic space within bacterial populations. Whole genome sequencing of the evolved resistant strains, identified key mutations in the 16s rRNA genes and the fusA gene encoding elongation factor-G, specific to antimicrobial agent gentamicin. Additional gene sequencing revealed parallel gentamicin resistant bacterial populations, evolved identical mutations within the fusA gene. Combined bioinformatic, phylogenetic and molecular docking analysis uncovered the biological significance of the fusA gene in the mutational pathways of gentamicin resistance in E. coli MG1655 in vitro. In contrast, the observed biological fitness costs associated with the acquisition of resistance conferring mutations, emphasised why such costly resistant genotypes were unidentified in natural and clinical settings. This study has established an experimental evolution model to explore the mutational pathways underlying antimicrobial resistance development in vitro. The ARGP offers a platform for the continued research into the acquisition of antimicrobial resistance through mutations, for more complex bacterial pathogens selected against a range of antimicrobial agents. As a tool, the ARGP can be utilised to inform therapeutic decisions based on the evolutionary risk management, provide new opportunities within the field of drug development and holds scope for its application within an educational setting.
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