MRC AMED - Systematic analysis of persistence mechanisms by high-throughput bar-seq and single cell analyses.

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Antimicrobial resistance (AMR) is a major global health issue which needs to be addressed by novel, collaborative research into the mechanisms of resistance. Persistence, occurs where a small, genetically-identical sub-fraction of a bacterial population that is exposed to antibiotic is, for unknown reasons, not killed. It is a feature of phenotypic (meaning non-genetic observable characteristic), rather than genetic variation and is very difficult to study because it occurs in a small sub-fraction of the population and is unstable. Yet persistence is the reason why antibiotics fail to cure many patients of disease. It may also be the progenitor of AMR, which is an urgent worldwide health issue. The UK team have expertise in analysing persister cells at the single-cell level, a requisite when observing this rare sub-population. The Japan team have complementary expertise in construction of a bacterial single-gene deletion library; the ASKA barcode deletion collection. Each mutant strain in the ASKA library has an artificial unique sequence that can be identified, via a technique called deep sequencing. The team have identified 13 mutants that exhibit increased persistence. The mechanisms involved are unknown and are often exhibited at the sub-population level, eg increased % of slow growers or aberrant morphology. This project will bring together the complementary expertise of the UK and Japan teams. The UK team will apply single cell imaging using microfluidics to perform computerized tracking of the bacterial mutants at the single cell and sub-population level to elucidate the role of differential morphology and growth rate in persistence. This knowledge could lead to development of novel drugs that target persister cell and thereby lead to higher cure rates, shorter treatment regimes and reduced AMR. The project will thereby contribute to the AMR global health threat.