Novel technologies to study single-cell response to environmental stimuli

Populations of genetically identical cells are not homogeneous, but contain subpopulations with phenotypic heterogeneity in their response to external stimuli. This has severely constrained our scientific understanding of key problems such as the emergence of drug resistance, where antimicrobial screening at the population level often leads to ambiguous findings, for instance due to the presence of drug-tolerant persister cells. Consequently, several key issues are not understood including drug uptake at the single-cell level, the effect of the drug on the single cell and the cell response, and phenotypic/genotypic differences among sub-populations of cells responding to stress in different ways. Addressing these issues using cutting-edge single-cell techniques is critical for an understanding of the dynamics of the response to drugs in heterogeneous populations and will allow both the more effective use of existing drugs and better ways of screening novel drugs.

The aim of this project is to investigate antibiotic tolerant bacteria that are responsible for recalcitrant microbial infections. The project will specifically focus on the accumulation of drugs in these cells and on how their efficacy is influenced by the specific nutritional environment around the cells. The PhD candidate, Ashley Smith, will use the Gram-negative bacterium Escherichia coli as an experimentally tractable model system; he will combine microbiology, microfluidics and microscopy approaches available in Biosciences at Exeter. The student will evaluate the influence of the nutritional environment on drug uptake in both antibiotic susceptible and antibiotic tolerant E. coli bacteria. These experiments will test the hypothesis that nutrient status in the environment predetermines membrane permeability and cell susceptibility to drugs.

Ashley will grow E. coli bacteria in microfluidic devices and create nutritional environments with limitations in carbon sources and amino acids that impose a stress on the cells. He will evaluate the influence of each nutritional environment on the formation of antibiotic tolerant bacteria to a variety of drugs including ampicillin, norfloxacin and gentamicin. He will thus gain knowledge of the nutritional environment(s) that favour the appearance of traits that are tolerant to specific drug(s).

By performing single-cell observations in microfluidic devices he will be able to characterize the physiology of antibiotic tolerant cells before the antibiotic challenge that is known to alter the tolerant phenotype at least at the transcriptomic level. Specifically, Ashley will quantify the growth rate and metabolic activity of antibiotic tolerant cells and he will compare these measurements with the ones on the cells, within the same clonal population, that are susceptible to the same dose of the antibiotic drug. He will quantify growth rate by using differential interference contrast microscopy and metabolic activity by measuring the fluorescent intensity of redox sensor green that is an indicator of bacterial reductase activity. These experiments will allow Ashley to gain information of the growth and metabolic state of antibiotic tolerant cells forming in a variety of nutritional states and shed light on the current controversy about dormancy in antibiotic tolerant cells. Ashley will also quantify drug uptake in, and efficacy against antibiotic tolerant cells forming in different nutritional environments. To this end, he will develop a protocol, based on drug autofluorescence, to quantify drug accumulation in single bacteria. These studies will test the hypothesis that nutrient status predetermines membrane permeability and cell susceptibility to drugs.

Collectively, these experimental efforts will provide optimal antibiotic treatments against antibiotic tolerant E. coli forming in a variety of nutritional environments and will be readily transferable to other Gram-negative bacteria of clinical interest.