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Title: A quantitative single-cell investigation of cell growth and antibiotic tolerance in Mycobacterium smegmatis
Author: Priestman, Miles
ISNI:       0000 0004 7658 4508
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Tuberculosis is a disease which has afflicted humans for millennia and currently holds the dubious title of the greatest cause of mortality by any single infectious agent. Mycobacterium tuberculosis-the causative agent of tuberculosis-is exquisitely adapted to the infection of humans, with the ability to persist within the hostile intracellular environment of the macrophage. To enable this, M. tuberculosis enacts a dormancy programme that permits long-term survival. This programme has been linked to the requirement for six months treatment to cure tuberculosis since it may enhance drug tolerance-the ability for bacteria to survive antibiotics without genetic resistance. In this thesis, I extensively utilise single-cell time-lapse microscopy coupled with microfluidics to observe Mycobacterium smegmatis cells over many generations. I describe in detail a robust methodology for long-term time-lapse and subsequent image analysis workflow, including its application to a challenging dataset. I then investigate how single cells maintain their cell size under diverse environmental conditions, and determine that birth length and division length are intimately linked by a fixed average extension when grown in standard medium-a so-called "adder". This relationship however breaks down when cells are grown in sub-optimal sole carbon sources. At the population level, I investigate antibiotic tolerance in M. smegmatis, and demonstrate that the ability to survive is highly enriched by growth phase, but not by nutrient deprivation. Finally, I describe an experimental platform with which the response of M. smegmatis to antibiotics can be observed at the single-cell level, and investigate factors that may predict the survival of M. smegmatis cells exposed to the anti-tubercular drug rifampicin, finding that single-cell growth rate and the ability to tolerate rifampicin are uncorrelated. Throughout this thesis, I use quantitative measures of single-cell properties that describe mycobacteria, data which may help develop novel strategies to combat the devastating global burden of tuberculosis.
Supervisor: Robertson, Brian ; Shahrezaei, Vahid Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral