Browsing by Author "Coetzee, Julian Luke"
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- ItemInvestigating the relation between persister formation and clinical outcome in Tuberculosis (TB) patients(Stellenbosch : Stellenbosch University, 2021-03) Coetzee, Julian Luke; Mouton, Jomien; Sampson, Samantha; Dippenaar, Anzaan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Despite progressive research regarding Mycobacterium tuberculosis, Tuberculosis (TB) still remains the top cause of mortality worldwide, with South Africa being considered one of the top ten TB burdened countries. Once infected with M. tuberculosis, TB disease can progress to an active disease state, or in the majority of cases, to an asymptomatic infection state known as latency or Latent TB infection (LTBI). LTBI has been associated with recurrent TB infection after a cured TB treatment outcome was achieved as individuals with LTBI are considered reservoirs of active M. tuberculosis. A subpopulation of bacteria known as persisters is thought to contribute to the LTBI state. Persisters are viable but non-replicating (VBNR) bacteria, which are recalcitrant to antibiotic treatment. There are major knowledge gaps regarding VBNR bacteria and their role in TB treatment outcome. Previously it was observed that patients who underwent TB treatment had remaining lesion activity post- treatment and presence of M. tuberculosis mRNA suggested the presence of unculturable bacteria likely being persisters. Based on positron emission tomography – computed tomography (PET/CT) scans patients were characterized as cured, recurrent or failed. In this study, we aimed to evaluate the correlation between persister formation and pulmonary TB (PTB) disease outcome. We exploited a dual fluorescence replication reporter plasmid, and assessed persister formation using a THP-1 infection model, which mimics the host environment pathogenic mycobacteria encounter upon infection. Whole genome sequencing (WGS) data of baseline and follow-up isolates was obtained to determine if isolates are genetically predisposed to persister formation. A total of eighteen baseline clinical M. tuberculosis isolates were selected for this study. Eight isolates represented bacteria from the cured patient group while ten isolates represented bacteria from the failed/recurrent patient group. Isolates were determined to be pure cultures and WGS data was obtained. In preparation for persister assay experiments, all eighteen isolates were transformed with the fluorescence dilution (FD) dual reporter plasmid pTiGc. Growth curves demonstrated that plasmid carriage had no impact on bacterial growth. The infection model enriched for persister-like cells as reflected by a subpopulation of VBNR bacteria. We found that all bacterial isolates possessed a level of replication heterogeneity at baseline both in vitro and intracellularly. Furthermore, isolates from the cured patients showed a significantly lower frequency of persister cells compared to that of isolates from the ailed/recurrent patient group. This suggests that the inherent tendency to form persister-like cells may have an impact on PTB treatment outcome. Data suggests that persister-like cell formation may be strain dependent. However, WGS data analysis were inconclusive. Furthermore, we recognize that the sample size is a crucial limiting factor in this study and further investigation with a larger cohort would be essential. This is the first study to use clinical strains of M. tuberculosis, obtained from failed/recurrent treatment outcome group, coupled with fluorescent reporters in combination with WGS data to investigate the relationship between persister formation and clinical outcome. Possible future work would be to to validate the phenotypic study findings in a murine model. Furthermore, future studies that determine the role of genetic variation in persister formation would greatly advance a patient-specific treatment regimen that could decrease the lengthy treatment duration.