AbstractTuberculosis (TB) is a bacterial disease that primarily affects the lungs and is caused by the bacillus Mycobacterium tuberculosis (M.tb). Approximately 1 in 3 people are latently infected globally, with 1.5 million people dying from active TB in 2015 alone. Prolonged therapy of at least six months with four antimicrobial drugs is required to successfully treat disease. This lengthy drug treatment is necessary to remove sub-populations of M.tb that are tolerant to antimicrobial drugs and as a result persist through drug therapy. Despite the hypothesised clinical significance of drug tolerant M.tb sub-populations in vivo, drug development models for TB fail to account for these complex mycobacterial populations.
Drug discovery models currently focus on planktonic bacterial growth systems that do not likely represent M.tb in vivo at which chemotherapy is targeted. The research presented in this thesis utilised a three-dimensional Rotary Cell Culture System (RCCS) to model mycobacterial growth in low shear conditions. Mycobacterium bovis (M. bovis) BCG was cultured in the RCCS optimised to induce biofilm formation, which was confirmed by scanning electron microscopy. M. bovis BCG biofilms were harvested after 21 days, homogenised to planktonic cell suspensions and antimicrobial susceptibility testing measured using culture, luminescent and colorimetric assays. Biofilm-derived bacilli were tolerant to isoniazid and streptomycin, a phenotype that could be rescued by passaging in
drug-free media. Transcriptional profiling of mycobacterial biofilms in comparison to stationary phase bacilli revealed that the stress response transcriptional regulators SigB and SigE were upregulated, as well as the ESX5 secretion system implicated in virulence. Isocitrate lyase, an essential glyoxylate shunt enzyme required for virulence in vivo and implicated in antimicrobial tolerance, was also induced in biofilm growth. Mammalian cell entry genes were downregulated, as well as dihydrofolate reductase, an enzyme required
for the activation of the anti-tuberculous drug pretomanid. Macrophages exposed to biofilm supernatants significantly induced TNFα but not IL-1β, suggesting a novel role for mycobacterial biofilms in host inflammatory responses. Finally, the role of a novel nuclease, nuc, was shown to not significantly increase rates of drug resistance in the laboratory strain
M.tb H37Rv. This work shows that low shear, rotary cell culture induces mycobacterial biofilm formation in vitro generating drug-tolerant bacteria that may more accurately represent in vivo drug-tolerant M.tb than traditional in vitro models. The results contribute novel research to the role of biofilm formation in TB, the clinical relevance of which is still not fully understood.
|Date of Award||May 2017|
|Supervisor||Simon Waddell (Supervisor) & Ian Cooper (Supervisor)|