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Title: Determining gene fitness in C. difficile in vitro and in vivo using high-throughput mutagenesis & transposon sequencing
Author: Fernandes, N. J.
ISNI:       0000 0004 8506 319X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2019
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Clostridium difficile is a Gram-positive, spore forming, anaerobic bacterium (Tool, Hall) and is the leading cause of antibiotic-associated diarrhoea in the world (Ghose, 2013). Little is known about the mechanisms by which C. difficile colonizes and persists within the gut environment. Classical mutagenesis tools are ineffective on C. difficile and the available methods of producing single deletion mutants are rather time consuming. Herein, we use high-throughput mutagenesis, combined with transposon-directed insertion site sequencing (TraDIS) to determine genes essential for survival under a variety of in vitro and in vivo conditions. High-density transposon mutagenesis was used to generate over 70,000 unique C. difficile mutants. Fitness of these mutants during their growth cycle was determined by TraDIS sequencing and analysis. The data helped elucidate genes essential for growth and sporulation in vitro. We have further exploited this method to assess gene fitness of a pool of C. difficile mutants in a mouse gut, thereby allowing us to gain insights into the infection and survival mechanisms of the bacterium. Our in vivo experiment has helped identify genes essential for colonisation, survival and pathogenesis of C. difficile and may even aid in identifying novel antimicrobial targets. A total of 62 in vivo datasets and 16 in vitro data sets have been analysed, making it one of the largest studies of its kind. Additionally, production of new libraries is underway to help decipher the mechanisms of the C. difficile S-layer, a component already showing promising results as an antimicrobial target. Cumulatively, the data derived has helped decipher the functionality of the C. difficile genome quicker and with greater ease. This progress will hopefully, bringing us one step closer to discovering novel treatments against C. difficile infection. The project has also given us a chance to improve methods of library generation and analysis. Future generation of larger datasets would enable us to improve accuracy and rate of data analysis.
Supervisor: Chaudhuri, R. R. ; Fagan, R. P. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available