Characterization of mycobacterium bovis persisters in South African wildlife

Ncube, Pamela (2020-12)

Thesis (MSc)--Stellenbosch University, 2020.

Thesis

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.

AFRIKAANS OPSOMMING: Mycobacterium tuberculosis infeksies in mense kan lei tot ‘n siekte toestand gepaard met simptome of onderdruk word en lei tot asimptomatiese latente infeksies. Dis onbekend of dieselfde gebeur in diere met Mycobacterium bovis infeksies. Ons siekte verstandhouding word gekompliseer deur die kennisgaping oor patogenesiteit en siekte progressie van M. bovis infeksies in verskeie diere. Die bestuur van latent geïnfekteerde diere kan verskil van dié met ‘n aktiewe siekte dus is dit belangrik om te evaulueer of M. bovis die potentiaal het om persister selle te vorm, die onderliggend oorsprong van latente infeksie. Die doel van die studie was om in vitro M. bovis persister vormasie te karakteriseer in suur kondisies wat die natuurlike makrofage omgewing naboots. ‘n Somtotaal van 23 M. bovis geïnfekteerde monsters van verskeie wildspesies was suksesvol gedekontamineer, gesuiwer en gegenotipeer. Spoligotipering het 5 verskillende M. bovis spoligotipes geïdentifiseer genaamd SB0121 (16 isolate), SB0130 (4 isolate), SB0140 (1 isolaat), SB1474 (1 isolaat), en SB1275 (1 isolaat). Ter voorbereiding van die persister vormings asseserings eksperiment is 22 van die 23 isolate suksesvol getransformeer met die fluoresserende pTiGc plasmied en groeikurwes het die gebrek aan groei defekte bevestig. Vyf verskillende M. bovis stamme was gekies om suur sensitiwiteit en persister vormasie te bestudeer deur die gebruik van ‘n suur-stres in vitro model. Die model verryk vir persister vormasie, uitgebeeld as ‘n sub-populasie van lewensvatbare maar geen- of stadig repliserende bakterieë. Beide laboratorium stamme, SAMMtb::pTiGc en BCG::pTiGc, het die hoogste gemiddelde persentasie persister selle gehad na 4 dae met 12.2 ± 1.5% (± SD) en 7.2 ± 0.6%, onderskeidelik. Die kliniese isolaat, PN18067_1::pTiGc, het die hoogste persentasie getoon van al die isolate met 1.3 ± 0.1%, van die hele populasie. Verder het PNMP20_1::pTiGc en PN18062_1::pTiGc beide ‘n gemiddeld van 0.2 ± 0.0% gehad. Die data dui daarop dat in suur gestresde omgewings: i) laboratorium stamme meer geneigd is om persister selle te vorm in vergelyking met kliniese isolate, ii) dat M. bovis persister selle kan vorm in suur toestande, iii) en dat persister vormasie verskil tussen stam genotipes. Dit is egter belangrik om in ag te neem dat die persentasie persister populasies onder dié spesifieke studie kondisies baie laag was (0.5 ± 0.6 %) en dalk nie ‘n noemenswaardige aanduiding kan wees van persister vormasie nie. Ons kon dus nie defnitiewe persister vormasie demonstreer nie, maar dis belangrik om in ag te neem dat ons net een stres kondisies bestudeer het by ‘n enekel tydspunt en slegs 3 kliniese isolate gebruik het. Dit los dus ons vraag van persister vormasie onopgelos en moet verder ondersoek word in die toekoms. Die studie is die eerste in sy soort wat kliniese isolate gekoppel met fluoresserende gene gebruik om die vermoë van M. bovis om persister selle te vorm bestudeer. Verder verskaf dit insig oor latente M. bovis infeksies in diere en verskaf bewys-van-konsep data vir toekomstige studies. Bevindings moet egter bekragtig word deur meer komplekse in vitro en in vivo modelle te gebruik in die toekoms asook addisionele kliniese isolate.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/110252
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