Investigating the drivers of gene amplification mediated resistance to beta-lactamase inhibitor combinations

Acquired genes conferring antimicrobial resistance in Gram-negative bacterial pathogens like Escherichia coli are frequently mobilized between bacterial cells by flanking transposon sequences, but these transposons can also drive gene duplications within the same strain under antimicrobial selection pressure.

These gene duplications are a key mechanism of resistance to beta-lactam/inhibitor combination antibiotics, which include the widely used piperacillin/tazobactam and most new antibiotics in the pre-clinical pipeline.

The overall aim of this study is to determine the key factors that influence the ability of a beta-lactamase gene /transposon combination to lead to gene amplification in response to antibiotic selection in Escherichia coli.

To achieve this, I will utilise my unique collection of E. coli with/without gene amplification of blaTEM-1, generate full length transposon sequences from their genomes and assemble using Gibson cloning.

This approach enables us to swap the narrow spectrum blaTEM-1 for broader spectrum beta-lactamases, to determine whether newer drug combinations are at risk to this resistance mechanism.

Transposon/AMR gene combinations will be cloned into a laboratory strain, and then 24 clinical isolates, and subsequent gene amplification and any effect on resistance and fitness will be measured.

This will allow us to examine the impact of transposon structure, AMR gene, and E. coli strain, on gene amplification, and enable improved prediction of this genotype from sequencing data. Objective 1.

Determine the reciprocal impact of transposon structure and AMR gene by testing different combinations for gene amplification under antibiotic selection Objective 2. Determine the impact of the genomic background on gene amplification