Browsing by Author "Parbhoo, Trisha"

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    Characterizing mycobacterium tuberculosis persister populations.
    (2021-09) Parbhoo, Trisha; Sampson, Samantha; Mouton, Jomien; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: The adaptation of Mycobacterium tuberculosis in the host contributes to tuberculosis (TB) being one of the leading pulmonary infectious diseases in the world. As infection with M. tuberculosis progresses, the bacilli experience various degrees of host stressors in the macrophage phagosome such as low pH, nutrient deprivation or exposure to toxic agents, which promotes cell-to-cell phenotypic variation. This contributes to the establishment of varying physiological states that may differentially drive disease progression. This includes a physiologically viable but non- or slowly replicating persister subpopulation, which was of interest in this study. Persisters remain metabolically active, and are characterized as multi-drug tolerant, despite the population being genetically susceptible to antibiotics. Persisters additionally evade the host immune response and macrophage antimicrobial processes by adapting their metabolic pathways to maintain survival and persistence in the host. Understanding of how host exposure promotes formation and survival of persisters is limited. Improved knowledge on the physiological state of M. tuberculosis persisters, including factors contributing to a persistent state is required to understand M. tuberculosis adaptation and survival in the host, in particular in the macrophage niche. In this study we applied a novel flow cytometry-based, culture-independent technique, termed fluorescence dilution (FD), to characterize the single-cell replication dynamics of M. tuberculosis following macrophage infection. Various fluorescent probes were tested in this study, highlighting specific challenges for application in M. tuberculosis. FD in combination with flourescent staining using a metabolic esterase reactive probe, calcein violet AM (CV-AM), provided an effective approach to investigate the physiological state of M. tuberculosis following macrophage infection. We assessed whether varying bacterial infection and intracellular burdens influence persister numbers inside macrophages. Phagocytosis and phagosome acidification was additionally inhibited using Cytochalasin D and Bafilomycin A1, respectively, to determine whether these host antimicrobial processes impact persister numbers. Limited or no replication contributes to the difficulty in identifying and recovering persisters as these bacteria are not accurately quantified by colony forming unit (CFU) plating. Developing a rapid flow cytometric method to enumerate and assess viability of M. smegmatis and M. tuberculosis in vitro and macrophage infection models has the potential to contribute to TB control programs. Conventional CFU counting methods are time consuming and hindered by the lengthy turn-around time (3-6 weeks), whereas flow cytometry sample acquisition, analyses and enumeration of M. tuberculosis could be attained within a few hours. Accurate counts were obtained from flow cytometric reference beads, eliminating the need for bacterial culturing.
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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.