Browsing by Author "Ncube, Pamela"

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    Characterization of mycobacterium bovis persisters in South African wildlife
    (Stellenbosch : Stellenbosch University, 2020-12) Ncube, Pamela; Sampson, Samantha Leigh; Miller, Michele Ann; Bagheri, Bahar; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.
    ENGLISH ABSTRACT: Mycobacterium tuberculosis infection in humans can either progress to disease, or in many patients, be contained and lead to a latent infection without evidence of active disease. However, it is unknown whether a similar scenario exists in animals infected with Mycobacterium bovis. Knowledge gaps in the pathogenesis and progression of disease in the wide spectrum of livestock and wildlife species that can be infected by M. bovis also complicate our understanding of this disease. Since management strategies may differ for latently infected animals, it is important to evaluate whether M. bovis has the potential to form persisters, which are believed to underlie latent infection. In this study, we aimed to characterize M. bovis persister formation upon in vitro acid stress which mimics the macrophage phagolysosome microenvironment. A total of 23 samples from naturally infected M. bovis wildlife species were successfully decontaminated, purified, and genotyped to the strain level. Spoligotyping identified a total of 5 different M. bovis spoligotypes, namely SB0121 (16 isolates), SB0130 (4 isolates), SB0140 (1 isolate), SB1474 (1 isolate), and SB1275 (1 isolate). In preparation for persister assay experiments, 22 of the 23 isolates were successfully transformed with the Fluorescent Dilution (FD) reporter plasmid pTiGc. Thereafter, growth curves confirmed that there were no growth defects of the transformants due to the carriage of the reporter plasmid. Five M. bovis strains were selected for acid sensitivity and persister assay experiments, using the acid stress in vitro model. This model enriches for persisters, as reflected by a sub-population of viable but non- or slowly replicating (VBNR) bacteria. Laboratory strains, SAMMtb::pTiGc and BCG::pTiGc, had the highest average percentage of VBNR cells at 12.2 ± 1.5% (± SD) and 7.2 ± 0.6%, respectively on day 4. In contrast, the clinical strain with the highest VBNR average percentage was PN18067_1::pTiGc at 1.3 ± 0.1%, while PNMP20_1::pTiGc and PN18062_1::pTiGc had a similar average percentage of 0.2 ± 0.0%. These data suggest that upon acid stress: (i) laboratory strains seem to have a higher propensity to form VBNR populations than three clinical isolates examined, (ii) M. bovis may demonstrate VBNR populations following acid stress, although these are very small, (iii) VBNR formation may vary depending on strain genotype. However, it is important to acknowledge that the VBNR populations detected under the conditions employed in this study were very small (0.5 ± 0.6 %), and may not be indicative of significant persister formation. We have, therefore, not definitively demonstrated persister formation, but note that we only investigated a single stressor, a single time point, and only 3 clinical isolates, leaving this an open question to be further investigated in the future. This is the first study to use clinical strains coupled with fluorescent reporters to investigate the ability of M. bovis to persist and provides some insights into a long-standing question on latent M. bovis in animals and describes proof of concept data for future investigation. Future work will need to validate these findings using more complex in vitro and in vivo models and additional clinical isolates.