High-resolution molecular typing of low-abundance multidrug resistant Enterobacterales in the human microbiota

Multidrug resistant Enterobacterales, including Escherichia coli and Klebsiella pneumoniae, are the leading cause of death associated with drug resistant bacterial infections globally. Drug resistant infections result in greater mortality, morbidity and higher cost to health systems, and disproportionally affect low and middle income countries (LMIC). For these pathogens, there is growing evidence that colonisation in the human gut typically precedes infection and exposure to antibiotics leads to amplification of drug resistant variants already present at low levels. Precisely where (hospital, community, environment) and how these bacteria are acquired, i.e. the route of transmission, is unclear and may differ between species. Understanding transmission is essential to informing infection prevention strategies aiming to interrupt transmission and reduce the number of infections. To do this, we need tools to accurately and cost effectively identify what pathogens are present in the human gut and how they change over time. Current gold standard for antimicrobial resistant (AMR) pathogen detection relies on selective culture from the original sample. This comes with some limitation: rare variants present at low level in the original sample may be missed, no information of the diversity of different stains as often only a single bacterial colony is further analysed and, it is not feasible for pathogens that cannot be cultured. Novel culture-independent, sequencing-based methods are emerging capable of addressing those limitations. Target-enrichment sequencing or metagenomic methods are promising tools with the potential to capture rare variants that are scarce in the original clinical sample, address strain diversity and work directly on extracted DNA without the requirement to culture. However, the most appropriate method to apply for E. coli and K. pneumoniae from clinical samples is unclear. In order to be useful in LMIC, cost effectiveness is key. The aim of this PhD project is to assess molecular target-enrichment methods on the performance to reliably, cost-effectively at scale, detect the low copy variants of E. coli and K. pneumoniae, antimicrobial resistance genes and the potential of strain-resolution directly from clinical samples. This project will leverage established microbiological workflows for DNA extraction from complex samples, in vitro spiked stool models, access to samples (stool samples/rectal swabs) from clinical settings in Liverpool and from Malawi with linked sequence information.