Understanding the pathway to multidrug resistant bacterial pathogens

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Infections caused by gram negative bacteria are among the most significant challenges in modern health care. The problem is exacerbated by a decline in antibiotic effectiveness and the emergence of new mechanisms of resistance. Mitigating this escalating problem depends upon detailed understanding of the genotypic and phenotypic changes that occur on the pathway to multidrug resistance (MDR). Widespread next-generation sequencing is now a routine component of pathogen surveillance and has confirmed the presence of antimicrobial resistance genes and MDR plasmids in global pandemic clones. However, little is known about the evolutionary 'stepping stones' that lead to the emergence of MDR. Understanding these could help with identifying early warning signs and public health interventions. We will investigate the underlying evolutionary mechanisms that give rise to MDR in bacteria. Unlike other studies, that focus on the presence of well known resistance genes - such as those linked to Extended-Spectrum Beta-Lactamase (ESBL) and Carbapenemase enzymes, we will identify the series of evolutionary events that underpin MDR acquisition. Specifically, we will investigate: (i) genes that confer low level resistance - the stepping stones to MDR; (ii) potentiating genes that may predispose certain strains to plasmid acquisition; (iii) accommodating genetic change that maintains MDR plasmids; (iv) if divergent strains have convergent evolutionary steps towards MDR; (v) the repeatability of MDR emergence. To address these question areas we will take an interdisciplinary approach, analysing very large diverse E. coli isolate collections. Combining comparative and functional genomics approaches with new bioinformatics methods and in vitro experimental evolution assays, we will characterize the evolutionary events and genetic changes that underpin the emergence and spread of pandemic MDR E. coli - the most common global cause of urinary tract infections and bacteraemia. First, we will identify genetic changes that covary with MDR. Second, we use genome-wide fitness profiling to investigate the genes and networks that potentiate or accommodate AMR phenotypes (tolerance, persistence etc.). Finally, we will investigate the reproducibility of evolution using an experimental evolution framework to test the repeatability of evolution in multiple genetic backgrounds of both clinical isolates and reference strains. This will identify predisposing factors and potentiating mutations that confer low level resistance and facilitating MDR plasmid acquisition and maintenance. This project will increase knowledge of the critical problem of MDR by identifying genetic changes underlying the emergence of resistance in pathogens responsible for some of the most common and serious systemic bacterial infections. Because we will characterize genetic changes in isolates from contemporary (real-world) infections, as well as across species diversity, we can be confident that our findings will be relevant to the ongoing battle against emergent MDR bacteria. In particular, helping design new laboratory testing for surveillance, outbreak risk models and targeted interventions.