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A quantitative single-cell investigation of cell growth and antibiotic tolerance in Mycobacterium smegmatis

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Title: A quantitative single-cell investigation of cell growth and antibiotic tolerance in Mycobacterium smegmatis
Authors: Priestman, Miles
Item Type: Thesis or dissertation
Abstract: 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.
Content Version: Open Access
Issue Date: Jun-2018
Date Awarded: Nov-2018
URI: http://hdl.handle.net/10044/1/65700
DOI: https://doi.org/10.25560/65700
Supervisor: Robertson, Brian
Shahrezaei, Vahid
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (Great Britain)
Funder's Grant Number: 1377289
Department: Medicine
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses

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