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

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.

The research from Japan have utilised the E. coli single-gene deletion library in which each deletion is marked with a unique ~ 20-nucleotide molecular artificial barcode (Otsuka et.al., 2015) to identify 13 persistence mutants. The mutant's competitive growth advantage or disadvantage was determined by pooling the mutants and analyzing the population in a mixed culture using high-throughput DNA sequencers following treatment with various antibiotics (i.e. persisters are often multi-drug tolerant). However, the persistence mechanism was not obvious in the majority of cases, with the average results most likely swamping the persister characteristics.

The UK team believe that by averaging results we often miss the exceptional, in this case persisters. The team will analyse the identify mutants at the single cell level. Time-lapse imaging of single cells will be carried out using a bespoke system consisting of a microfluidics platform and a Nikon confocal microscope equipped with an environmental chamber, motorized stage and perfect focus system, with image scans taken every minute. The experiments for persister cell discovery will consist of grow, kill, regrow and rekill stages. This final step is often left out in persister analysis and is a requisite to determining phenotypic resistance, a key characteristic of persistence. Computerised tracking will the be utilised to determine any difference in length at birth, division, elongation rate, division time as described in detail (Hu et al., 2017). Morphology will also be observed. Alongside, both teams will also be constructing fluorescent reporter strains for each of the mutants which will also allow the localisation of gene product at the single cell level. The majority of persistence mechanisms occur at the sub-population level and this research will identify novel persistence and AMR mechanisms, the characterization of which would be integral in combating this global health threat.

The discoveries of this exciting and important research project will have a number of clear and demonstrable impacts on wide ranging beneficiaries. The impact will be regularly monitored and assessed by the applicants.


1.The UK, Japan, and world-wide population in general. The availability of improved drugs with which to combat this global health threat would benefit the global population. There would be less disease and less dissemination of disease, which can be linked to a huge benefit in the terms of livelihood and economic benefit and more importantly to survival itself.


2.The healthcare system in the UK, Japan and world-wide. This will include the clinicians involved in the healthcare system. Reducing treatment time and improving efficacy would dramatically benefit the healthcare system both here and abroad, thus increasing its effectiveness.


3.Pharmaceutical companies, such as GSK, involved in drug development and the screening of potential drugs to combat bacterial persistence.


4.The press, media and public. The University of Surrey has an active media centre (http://www.surrey.ac.uk/mediacentre/press/index.htm), which will be utilized to promote scientific breakthroughs associated with this proposal. In addition, drug-tolerant persisters are a signature of most pathogens and as such these findings may be applied to many more diseases, thus further increasing the impact of this work. NAIST also has a public relations department that will be involved.