Convergent evolution of Enterobacteriaceae in epidemiological networks with high antimicrobial use

Disease-causing bacteria that are resistant to antimicrobials are a global health concern. While there are many research programs looking into the nature and spread of antimicrobial resistance (AMR), little is known about the evolutionary 'stepping stones' that precede the emergence of resistance. Understanding these could help with identifying early warning signs and targeting public health interventions.

We will investigate the underlying evolutionary mechanisms that give rise to antimicrobial resistance in bacteria. Unlike other studies, that are done solely in the laboratory, our novel approach will do this using a unique, real-world situation of bacterial populations transmitting among a high antimicrobial use community. We will focus on enteric bacteria, which cause diarrhoeal disease and are pathogens with the most worrying AMR.

We will study bacterial genome sequences from real infections and identify genetic changes that happen alongside the development of AMR using the latest bioinformatic methods. We will then analyse hundreds of bacterial strains in the laboratory looking at behaviours that might help the bacteria develop AMR and associate these behaviours with our genetic changes, developing models to quantify which of these signatures is most important. Finally, we will confirm that these genetic changes contribute to the development of AMR by evolving bacteria with and without the genetic change in the presence of antimicrobials.

This work will increase our knowledge of the critical problem of AMR by identifying genetic changes underlying the emergence of AMR in an important bacterial group. Because we are finding the genetic changes in a real-world setting and testing our hypotheses across hundreds of different bacterial strains, we know our findings will be important and could help us prevent AMR emergence by, for example, helping design new laboratory testing for use in AMR surveillance.

AMR emergence is a global concern which we must work to prevent. Our proposal will identify novel genetic and phenotypic signatures that precede and promote the emergence of AMR in enteric organisms. We work with a recently emerged, highly relevant real-world epidemiological scenario; the parallel emergence of multiple highly AMR enteric organisms sexually transmitting among UK men who have sex with men. Our pilot work on a subset of Shigella epidemics revealed previously uncharacterised genetic signatures associated with the emergence of AMR in this setting.

We will expand this pilot work with extensive routinely generated data from an up-to-date collection of phylogenetically diverse Enterobacteriaceae and employ multiple GWAS methodologies to comprehensively identify genetic signatures that accompany AMR emergence in this setting. We will then statistically associate these genetic signatures with AMR-associated phenotypes (e.g. tolerance, persistence) using our novel translatable approach 'bulk phenotyping of epidemiological replicates' and quantitate the contribution of the genetic signatures to AMR emergence. To validate the causal role of these candidates in AMR emergence, we will combine molecular microbiology with experimental evolution, reconstructing the genetic signatures in novel and model backgrounds and compare their adaptation to a high antimicrobial environment, allowing for AMR development by both de novo mutation and horizontal gene transfer.

This work will increase our knowledge of the critical problem of AMR by identifying genetic signatures underlying its emergence in an important pathogen group. Our approach of drawing the genetic signatures from a real epidemiological scenario, and supporting them through phenotyping of hundreds of clinical isolates mean our findings will be highly translatable to real-world applications such as enhancing phenotypic and genotypic surveillance programs for enteric bacteria in public health.