Development and characterisation of a low-shear mycobacterial biofilm model

  • Daire Cantillon

Student thesis: Doctoral Thesis

Abstract

Tuberculosis (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 AwardMay 2017
LanguageEnglish
Awarding Institution
  • University of Brighton
SupervisorSimon Waddell (Supervisor)

Cite this

Development and characterisation of a low-shear mycobacterial biofilm model
Cantillon, D. (Author). May 2017

Student thesis: Doctoral Thesis