Browsing by Author "Pule, Caroline"

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    Deciphering the physiology of drug tolerant and resistant Mycobacterium tuberculosis
    (Stellenbosch : Stellenbosch University, 2021-03) Pule, Caroline; Sampson, Samantha Leigh; Louw, Gail Erika; Warren, Robin Mark; Mouton, Jacoba Martina; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Poor adherence to treatment for tuberculosis (TB) disease and the rising incidents of drug resistant Mycobacterium tuberculosis strains are factors that negatively influence TB control. The current study was designed to explore some of the key knowledge gaps concerning M. tuberculosis physiology; looking at the effect of the diverse M. tuberculosis genetic backgrounds and the presence of ropB mutation on the transcriptome, looking at M. tuberculosis host response and the likelihood as to whether induced mycobacterial tolerance can provide a reservoir from which genetic resistance can arise. Exploring some of these key knowledge gaps was imperative, given the fact that, lengthy anti-TB drug treatment could be required to entirely eradicate some of M. tuberculosis strains, and non-compliance with completing treatment might lead to the emergence of multidrug (MDR)-TB. Firstly, we investigated the effect of M. tuberculosis strains with different genetic backgrounds on their total transcriptomic profiles (as a proxy for the physiological state). Secondly, we examined the influence of rpoB Ser531Leu mutation and the effect of isoniazid (INH) treatment (24h) at sub-lethal concentrations on the transcriptomic profiles of rifampicin (RIF)-resistant (K636RIF) and susceptible (K636WT and H37RVWT) M. tuberculosis strains, using RNA-sequencing (RNA-Seq) and Real-Time quantitative polymerase chain reaction (RT-qPCR) techniques. RNA-Seq analysis identified significantly differentially expressed genes in the transcriptomes of K636WT, H37RVWT and K636RIF M. tuberculosis strains. Our comparative transcriptomic data of K636WT relative to H37RvWT M. tuberculosis strains demonstrated that different genetic backgrounds influenced the total transcriptome. We demonstrated that rpoB Ser531Leu mutation has an impact on the transcriptional responses of K636WT relative to K636RIF M. tuberculosis strains. Our data did not demonstrate an effect of INH treatment on the transcriptomes of M. tuberculosis strains from different genetic backgrounds, making this one of our limitations. We then assessed the host immune response after infection with RIF-resistant (K636RIF and H37RvRIF) and susceptible (K636WT and H37RVWT) M. tuberculosis strains using the luminex x multi-analyte profiling (xMAP) technology and enzyme-linked immunosorbent assay (ELISA). Our host response data (Chapter 4) revealed no differences in host response to K636WT and H37RvWT M. tuberculosis strains from different genetic backgrounds. In contrast, there were differences in host response to K636WT and K636RIF M. tuberculosis strains in a RAW264.7 macrophage model of infection. This was confirmed by the observed varying secretion levels of cytokines and chemokines (IL-6, IL-12p40 and RANTES) required to mediate M. tuberculosis growth and survival after 24 - 48h of infection. We further investigated whether viable but non-replicating (VBNR) persisters Mycobacterium smegmatis sub-populations, when exposed to high INH concentrations, may provide a pool from which genetic resistant mutants can arise. We used a combination of a fluorescence dilution (FD) reporter system, flow cytometry and fluorescence-activated cell sorting (FACS) to detect, quantify and separate VBNR and actively replicating (AR) M. smegmatis bacterial populations following INH treatment. Subsequently, we performed PCR to amplify the katG and inhA promoter and Sanger sequencing to identify mutations in these genes that are commonly associated with INH resistance. Our flow cytometry results showed successful detection of VBNR and AR bacterial populations in M. smegmatis::pTiGc following INH pre-treatment at high concentration (30x MIC) for 72h. Mutation frequencies of different sorted populations were determined as 3.51% for M. smegmatisVBNR, 5.20% for M. smegmatisAR and 0.02% for M. smegmatis UNT. Sanger sequencing data demonstrated a high percentage of mutations in the inhA promoter (C-15T) (76% in VBNR; 64% in AR) compared to mutations in the katG gene (48% in VBNR; 44% in AR). However, the difference was not statistically significant (p > 0.1). This study addressed the following knowledge gaps: it advanced our understanding about the M. tuberculosis physiology. It confirmed that strain genetic background and the presence of rpoB Ser531Leu mutation may play a role in the physiological state of M. tuberculosis strains as reflected in their transcriptomes. It confirmed that host response in vitro is influenced by M. tuberculosis strain genotype and that infection with K636WT, H37RvWT and H37RvRIF M. tuberculosis strains will result in the secretion of pro-inflammatory cytokines and chemokines while infection with K636RIF M. tuberculosis strain (with rpoB Ser531Leu mutation) might induce secretion of anti-inflammatory cytokines (second line of host defense). This study was the first to successfully use a FACS analysis in combination with the FD reporter system to detect, isolate and quantify VBNR from AR M. smegmatis, following INH pre-treatment at high concentrations. We speculate that our results showed that the VBNR persisters‟ sub-population is likely to provide a reservoir from which genetic resistant mutants can arise, when treated with high INH concentrations as made evident by the observed INH resistant mutants in VBNR M. smegmatis. This work contributed further knowledge to finding better strategies to prevent the spread of emerging MDR, as well as extensively drug resistant M. tuberculosis.
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    Defining the role of efflux pump inhibitors on anti-TB drugs in Rifampicin resistant clinical Mycobacterium Tuberculosis isolates
    (Stellenbosch : Stellenbosch University, 2014-04) Pule, Caroline; Victor, T. C.; Louw, G. E.; Warren, R. M.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences, Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Central dogma suggests that mutations in target genes is the primary cause of resistance to first and second-line anti-TB drugs in Mycobacterium tuberculosis. However, it was previously reported that approximately 5% of Rifampicin mono-resistant clinical M. tuberculosis did not harbor mutations in the rpoB gene. The present study hypothesized that active efflux plays a contributory role in the level of intrinsic resistance to different anti-TB drugs (Isoniazid, Ethionamide, Pyrazinamide, Ethambutol, Ofloxacin, Moxifloxacin, Ciprofloxacin, Streptomycin, Amikacin and Capreomycin in RIF mono-resistant clinical M. tuberculosis isolates with a rpoB531 (Ser-Leu) mutation. This study aimed to define the role of Efflux pump inhibitors (verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine) in enhancing the susceptibility to different anti-TB drugs in the RIF mono-resistant clinical isolates. The isolates were characterized by determining the level of intrinsic resistance to structurally related/unrelated anti-TB drugs; determining the effect of EPIs on the level of intrinsic resistance in the isolates and comparing the synergistic properties of the combination of EPIs and anti-TB drugs. To achieve this, genetic characterization was done by PCR and DNA sequencing. Phenotyping was done by the MGIT 960 system EpiCenter software to determine the MICs of the different anti-TB drugs and the effect of verapamil and carbonylcyanide m-chlorophenylhydrazone on determined MICs. Due to inability to test reserpine in a MGIT, a different technique (broth microdilution) was used for the reserpine experiment. Additionally; fractional inhibitory concentrations (FIC) indices were calculated for each of these drugs. The FIC assess the anti-TB drugs/inhibitor interactions. STATISTICA Software: version 11 was used for statistical analysis. Results revealed that the RIF mono-resistant isolates were sensitive at the critical concentrations of all 10 drugs tested, with the exception of Pyrazinamide. This could be explained by the technical challenges of phenotypic Pyrazinamide testing. A significant growth inhibitory effect was observed between the combination of EPI and anti-TB drug exposure in vitro. This suggests that verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine play a significant role in restoring the susceptibility (decrease in intrinsic resistance level) of the RIF mono-resistant isolates to all anti-TB drugs under investigation. Additionally, a synergistic effect was observed by the combination treatment of the anti-TB drugs with the different EPIs. Based on these findings, we proposed a model suggesting that efflux pumps are activated by the presence of anti-TB drugs. The activated pumps extrude multiple or specific anti-TB drugs out of the cell, this in turn decrease the intracellular drug concentration, thereby causing resistance to various anti-TB drugs. In contrast, the addition of EPIs inhibits efflux pump activity, leading to an increase in the intracellular drug concentration and ultimate cell death. This is the first study to investigate the effect of different efflux pumps inhibitors on the level of intrinsic resistance to a broad spectrum of anti-TB drugs in drug resistant M. tuberculosis clinical isolates from different genetic backgrounds. The findings are of clinical significance as the combination of treatment with EPI and anti-TB drugs or use of EPIs as adjunctives could improve MDR-TB therapy outcome.