Browsing by Author "Parbhoo, Trisha"

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    Characterizing Mycobacterium tuberculosis persister populations
    (2021-09-20) Parbhoo, Trisha; Sampson, Samantha; Mouton, Jomien
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    Recent developments in the application of flow cytometry to advance our understanding of Mycobacterium tuberculosis physiology and pathogenesis
    (International Society for Advancement of Cytometry, 2020-05-21) Parbhoo, Trisha; Sampson, Samantha L.; Mouton, Jacoba M.
    The ability of the bacterial pathogen Mycobacterium tuberculosis to adapt and survive within human cells to disseminate to other individuals and cause active disease is poorly understood. Research supports that as M. tuberculosis adapts to stressors encountered in the host, it exhibits variable physiological and metabolic states that are time and niche-dependent. Challenges associated with effective treatment and eradication of tuberculosis (TB) are in part attributed to our lack of understanding of these different mycobacterial phenotypes. This is mainly due to a lack of suitable tools to effectively identify/detect heterogeneous bacterial populations, which may include small, difficult-to-culture subpopulations. Importantly, flow cytometry allows rapid and affordable multiparametric measurements of physical and chemical characteristics of single cells, without the need to preculture cells. Here, we summarize current knowledge of flow cytometry applications that have advanced our understanding of the physiology of M. tuberculosis during TB disease. Specifically, we review how host-associated stressors influence bacterial characteristics such as metabolic activity, membrane potential, redox status and the mycobacterial cell wall. Further, we highlight that flow cytometry offers unprecedented opportunities for insight into bacterial population heterogeneity, which is increasingly appreciated as an important determinant of disease outcome. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
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    Using fluorescene to understand Mycobacterial Heterogeneity at a Single Cell Level
    (Stellenbosch : Stellenbosch University, 2017-03) Parbhoo, Trisha; Mouton, Jomien; Sampson, Samantha; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Tuberculosis remains a major worldwide health threat. Among those infected there is risk of Mycobacterium tuberculosis, the causative agent of tuberculosis, developing into an asymptomatic dormant state. Cell-to-cell phenotypic variation is known to contribute to the establishment of diverse colonization states to evade host immune responses. There are major knowledge gaps regarding dormant or viable but non-culturable (VBNC) bacteria as they are difficult to isolate and heterogeneous populations are not reflected by colony forming unit plating. This has led to the application of single-cell techniques, such as flow cytometry, which offers a rapid, high-throughput tool to analyse the physiological and biochemical characteristics of bacteria at a single cell level in M. tuberculosis. Here we discussed various applications of flow cytometry for further understanding the physiological nature of bacterial systems. We aim to provide a better understanding of these physiological states of mycobacteria, and this thesis describes efforts to develop tools towards this aim. To identify and enumerate live and dead Mycobacterium smegmatis within a heterogeneous population we optimised the LIVE/DEAD BacLight Bacterial Viability and counting kit, exploiting flow cytometry. M. smegmatis was quantified by means of standardization beads, and the kit shows promise for developing a rapid, culture-free counting method for mycobacteria, ultimately replacing colony forming unit (CFU) plating. Efforts were initiated for applying this method to bacteria exposed to various anti-tuberculosis antibiotics, as well as for bacteria spiked into artificial sputum, which will require further investigation. In addition, flow cytometry was applied together with a recently developed Fluorescence Dilution (FD) reporter system, to enable measurement of differentially replicating mycobacteria in macrophage infection models. Previous studies have reported M. smegmatis to be rapidly killed upon uptake into macrophages. In contrast, results from our lab using a M. smegmatis macrophage infection model uncovered an unexpected sub-population of apparently dividing M. smegmatis. The nature of this population was explored using a fluorescently labelled anti-tuberculosis cell-surface binding antibody in combination with FD, for determination of whether the replicating population was intra- or extracellular of the macrophage. However, further investigation will need to be performed to determine the location of this population. The findings of this study suggest the feasibility of a real-time tool to distinguish and enumerate live and dead cells within a heterogeneous mycobacterial population. Additionally, FD in combination with other markers offers a promising technique for studying population-wide adaptation to environmental stress, or even to anti-tuberculosis drugs.