Masters Degrees (Molecular Biology and Human Genetics)

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Browsing Masters Degrees (Molecular Biology and Human Genetics) by Subject "Antibacterial agents"

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    Characterising persister growth in response to environmental conditions using a biofilm model
    (Stellenbosch : Stellenbosch University, 2023-03) Mathee, Raadhiyah; Sampson, Samantha Leigh; Mouton, Jomien; Bagheri, Bahareh; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: The rise in antibiotic therapy failure worldwide poses a serious threat to successful tuberculosis (TB) treatment. This is partly due to a subpopulation of cells known as persisters. Persisters possess the ability to switch to a temporary drug tolerant state, avoiding the action of antimicrobials thereby contributing to the lengthy TB treatment regimen and high treatment failure rates. Persister formation is thought to be influenced by various environmental conditions. Biofilms offer a model system to study persister formation and physiology since cells harboured within biofilms are exposed to heterogeneous environments with varying levels of oxygen and nutrients. Moreover, biofilms are enriched for persister cells and can withstand antibiotic treatment and can thus serve as an appropriate model to study the nature of persisters. Previously the isolation and characterisation of these persisters proved to be challenging using standard culture techniques, slowing the progress in research. However, with the development and validation of techniques using fluorescence dilution and flow cytometry, we are now offered insight into the microbial lifestyle at a single cell level. The fluorescence dilution reporter system carries a constitutive green fluorescent protein used as a marker of viability and an inducible red fluorescent protein used to monitor bacterial replication. We attempted to utilise a hydrodynamic flow cell system as an in vitro biofilm model to investigate bacterial growth and replication dynamics. However, further use of this system was abandoned as bacteria failed to adhere to the substratum, preventing further applications. The widely used microtiter plate assay was then employed to carry out all objectives of this study. Protocol modification and optimisation were performed to incorporate the use of the reporter strain, and to remove and disperse the cells. Results showed changes in cell morphology after exposure to biofilm microenvironments, particularly the presence of shorter rods and more circular shapes. The bacterial replication dynamics of biofilm cell populations revealed a subpopulation of cells that retained high levels of red fluorescent intensity, potentially indicative of persisters/persister like cells. We then explored spatial growth patterns in biofilms to determine cell localisation in response to induction using Confocal Laser Scanning Microscopy. This provided overall mapping of the pellicle structure and showed evidence of individual cells expressing red fluorescent intensity. The findings of this study provide insight into the use of a biofilm model to study persisters by Imaging Flow Cytometry and a starting point for further study incorporating quantitative assessment of intact biofilm structures.
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    Investigating the host-directed therapeutic potential of curdlan-functionalised PLGA nanoparticles in the treatment of intracellular Mycobacterium tuberculosis
    (Stellenbosch : Stellenbosch University, 2020-03) Du Plessis, Su-Mari; Sampson, Samantha; Dube, Admire; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Despite the availability of antibacterial agents, tuberculosis (TB) remains the leading cause of death by a single infectious agent globally, accounting for millions of deaths annually. The high mortality and morbidity rates are associated with antibiotic resistance and tolerance. Further, Mycobacterium tuberculosis, the causative agent of TB, has successfully evolved various strategies to evade an antibacterial immune response. M. tuberculosis infection is a continuous interaction between the host and the bacterium. An improved fundamental understanding of how this bacterium can survive within host immune cells has paved the way for the development of host directed therapies (HDT) that utilize a different approach to conventional antibiotics; and such strategies are desperately required for the eradication of TB. HDT is an approach targeted at the host immune system instead of M. tuberculosis itself. By targeting the host immune system, there is potential to restrict the possible acquisition of genetic resistance, since the bacteria itself is not the target. To favour a protective immune response at the molecular level, immunotherapeutic nanoparticles (NPs) are being exploited. NPs are particulate structures at the nanoscale (1 – 1000 nm) with the ability to target specific cells, with such targeting achieved through modification of the surface of the NP with bioactive ligands. By targeting specific cells, the NPs can localize at the pathogen infected site and elicit an antibacterial response through ligand-receptor interactions. In this study, to target M. tuberculosis-infected macrophages (primary host immune cells), poly(lactide- co-glycolide) (PLGA) NPs were functionalised with curdlan, an immune-stimulatory polysaccharide known to target the dectin-1 receptor expressed on macrophages. Curdlan-dectin-1 interactions are known to stimulate the NF-kβ pathway that produces bactericidal factors such as oxidative species and pro-inflammatory cytokines and activate downstream pathways capable of killing intracellular bacilli. Thus, it is hypothesized that the introduction of curdlan-functionalised PLGA (C-PLGA) NPs to M. tuberculosis infected macrophages will stimulate the cells, leading to the death of the intracellular bacilli. Four different formulations were synthesized containing different curdlan loads and these included PLGA, 2%, 5% and 8% w/w C-PLGA NPs. Dynamic light scattering data confirmed the particles to be in the nano-size range with a negative zeta potential. The cytotoxicity of the NPs towards RAW264.7 macrophages was assessed using the MTT assay over 72h. Macrophages infected with M. tuberculosis ΔleuDΔpanD::pMV306hsp+lux expressing the bacterial luciferase gene, were used to assess the killing efficacy of the NPs (using luminescence as a proxy for cell numbers, with confirmatory colony forming unit (CFU) plating). While CFU data showed a trend towards reduction in M. tuberculosis bacterial numbers compared to untreated control following NP treatment, interpretation of results was cofounded by possible macrophage lysis, and this will require further investigation. Cytokine quantification highlighted the immune stimulating capabilities of the curdlan-functionalised NPs and upregulation of TNF-α was detected after 72h. The findings of this study support the hypothesis that C-PLGA NPs can stimulate M. tuberculosis infected macrophages, potentially leading to death of intracellular M. tuberculosis. This demonstrates the potential of C-PLGA NPs to be further developed as a HDT for TB.