Browsing by Author "Mathee, Raadhiyah"

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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.