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Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing
Sims-Handcock, Jethro Dylan. 2023. Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing. Unpublished masters dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/4632784b-6f08-4...
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867613856239452160 |
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| access_status_str | Open Access |
| author | Sims-Handcock, Jethro Dylan |
| author2 | Streicher, Elizabeth |
| author_browse | Sims-Handcock, Jethro Dylan Streicher, Elizabeth |
| author_facet | Streicher, Elizabeth Sims-Handcock, Jethro Dylan |
| author_sort | Sims-Handcock, Jethro Dylan |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Sims-Handcock, Jethro Dylan. 2023. Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing. Unpublished masters dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/4632784b-6f08-4ee8-9035-133392bda700 |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/128493 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:42:46.825Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/128493 Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing Sims-Handcock, Jethro Dylan Streicher, Elizabeth Kriel, Nastassja Loubser, Johannes Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics. Antitubercular agents Human genetics -- Variation Multidrug resistance Tuberculosis -- Alternative treatment UCTD Sims-Handcock, Jethro Dylan. 2023. Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing. Unpublished masters dissertation. Stellenbosch : Stellenbosch University [online]. Available at: https://scholar.sun.ac.za/items/4632784b-6f08-4ee8-9035-133392bda700 Thesis (MSc)--Stellenbosch University, 2023. ENGLISH ABSTRACT: Multi-Drug Resistant tuberculosis (MDR-TB) remains a public health crisis with infections requiring expensive and intensive treatments. Outbreaks therefore represent significant threats to global health security and understanding them remains crucial to their mitigation and control. Understanding the genetic variants involved in their resistance is essential for the development of more effective treatment strategies and novel therapeutics. In this study, an X-family MDR-TB outbreak originating in the West Coast district of South Africa was described preliminarily using spoligotyping and sanger sequencing-mediated drug resistance predictions. This was followed by whole genome sequencing (WGS) to fully differentiate the MDR isolates and establish the phylogenetic relationships of the outbreak. WGS data was processed using the in-house Universal Sequence Alignment Pipeline (USAP) while genomic drug susceptibility predictions were done using the TB-profiler bioinformatic pipeline. Novel genomic variants were identified in genes linked to the Isoniazid (INH) resistance phenotype and were selected for further functional investigation. The variants C171G rv0024 (NLPC/P60 family protein), T1147C rv0343 (INH inducible protein C), T764C rv0482 (murB) and G205C rv2333c (stp drug efflux pump) were selected for in silico structural predictions. The variant proteins and their wild type counterparts were cloned into the mycobacterial plasmid, pFLAG. The constitutive over-expressing plasmids were transformed into the model organism Mycobacterium smegmatis to functionally investigate the genes contribution to phenotypic INH resistance and the impact on mycobacterial fitness. Minimum inhibitory concentrations were measured using a resazurin microplate assay (REMA) while fitness was assessed with exponential phase limited growth curves. All transformed strains showed lower MIC values than that of the wild type M. smegmatis, likely due to the additional resource costs imposed by the expression of recombinant proteins. The T1147C iniC variant had a 2 fold higher MIC range than the WT iniC and positive control, indicative of a more active role in INH resistance than previously demonstrated. Generally the variant protein transformants also induced depressed growth rates compared to their WT counterparts and to controls. The identified target genes and the identified variants still represent potentially significant mutations and should be further investigated in Mycobacterium tuberculosis. This study also serves as a model for WGS investigations into the effects of novel variants on fitness and their contributions to the drug resistance phenotype, as well as to highlight targets for future adjunctive treatments. AFRIKAANSE OPSOMMING: Multi-Weerstandige Tuberkulose (MDR-TB) is 'n openbare gesondheidskrisis met infeksies wat duur en intensiewe behandelings vereis. Uitbrake van MDR_TB verteenwoordig dus beduidende bedreigings vir wêreldwye gesondheidsekuriteit en om dit te verstaan is noodsaaklik om dit te verlig en te beheer. Om die genetiese variante betrokke by TB weerstandigheid te verstaan, is noodsaaklik vir die ontwikkeling van meer effektiewe behandelingstrategieë en nuwe terapeutika. In hierdie studie is 'n Xfamilie MDR-TB-uitbraak wat in die Weskus-distrik van Suid-Afrika ontstaan het, voorlopig beskryf deur gebruik te maak van spoligotipering en sanger-volgordebepaling-gemedieerde middelweerstandvoorspellings. Dit is gevolg deur heelgenoomvolgordebepaling (WGS) om die MDRisolate ten volle te onderskei en die genetiese verwantskappe van die uitbraak vas te stel. WGS-data is verwerk deur gebruik te maak van die plaaslik ontwikkelde Universal Sequence Alignment Pipeline (USAP) terwyl genomiese weerstandigheids voorspellings gedoen is met behulp van die TB-profiler bioinformatiese pyplyn. Nuwe genomiese variante is geïdentifiseer in gene gekoppel aan die Isoniazid (INH) weerstand fenotipe en is geselekteer vir verdere funksionele ondersoek. Die variante C171G rv0024 (NLPC/P60 familie proteïen), T1147C rv0343 (INH induseerbare proteïen C), T764C rv0482 (murB) en G205C rv2333c (stp middel uitvloeipomp) is geselekteer vir in silico strukturele voorspellings. Die variante proteïene en hul wildtipe eweknieë is in die mikobakteriese plasmied, pFLAG, gekloneer. Die konstitutiewe ooruitdrukkingsplasmiede is getransformeer in die model organisme Mycobacterium smegmatis om die gene se bydrae tot fenotipiese INH weerstand en die impak op bakteriële fiksheid funksioneel te ondersoek. Minimum inhiberende konsentrasies is gemeet met behulp van 'n resazurin mikroplaattoets (REMA) terwyl fiksheid geassesseer is met eksponensiële fase beperkte groeikurwes. Alle getransformeerde stamme het laer MIC waardes getoon as dié van die wilde tipe M. smegmatis, waarskynlik as gevolg van die bykomende hulpbronkoste wat deur die uitdrukking van rekombinante proteïene opgelê word. Die T1147C iniC-variant het 'n 2 maal hoër MIC-reeks as die WT iniC en positiewe kontrole gehad, wat 'n meer aktiewe rol in INH-weerstand aandui as wat voorheen gedemonstreer is. Oor die algemeen het die variante proteïene ook verlaagde groeitempo's vertoon in vergelyking met hul WT-eweknieë en kontroles. Die geïdentifiseerde teikengene en die geïdentifiseerde variante verteenwoordig steeds potensieel beduidende mutasies en moet verder ondersoek word in Mycobacterium tuberculosis. Hierdie studie dien as 'n model vir WGS-ondersoeke na die uitwerking van nuwe variante op fiksheid en hul bydraes tot die middelweerstandfenotipe, asook om teikens vir toekomstige aanvullende behandelings uit te wys. Masters 2023-02-26T17:11:21Z 2023-08-30T13:08:13Z 2023-04-01 2023-02-26T17:11:21Z 2023-08-31T09:18:52Z 2023-02-26T17:11:21Z 2023-08-31T09:18:52Z 2023-03 Thesis https://scholar.sun.ac.za/handle/10019.1/128493 en Stellenbosch University application/pdf 125 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Antitubercular agents Human genetics -- Variation Multidrug resistance Tuberculosis -- Alternative treatment UCTD Sims-Handcock, Jethro Dylan Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing |
| title | Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing |
| title_full | Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing |
| title_fullStr | Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing |
| title_full_unstemmed | Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing |
| title_short | Investigating novel variants associated with the isoniazid resistance phenotype in MDR-TB using whole genome sequencing |
| title_sort | investigating novel variants associated with the isoniazid resistance phenotype in mdr tb using whole genome sequencing |
| topic | Antitubercular agents Human genetics -- Variation Multidrug resistance Tuberculosis -- Alternative treatment UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/128493 |
| work_keys_str_mv | AT simshandcockjethrodylan investigatingnovelvariantsassociatedwiththeisoniazidresistancephenotypeinmdrtbusingwholegenomesequencing |