whole-genome-deoxyribonucleic-acid-extraction-from-mycobacterium

Whole-Genome Deoxyribonucleic Acid Extraction from <em>Mycobacterium</em> Species <em>via</em> the Cetyltrimethylammonium Bromide Technique

Efficient and reliable DNA extraction is a fundamental step in molecular biology, providing high-quality genetic material for downstream applications such ...

Stellenbosch University

Non-tuberculous mycobacteria (NTM) infections are increasing in incidence and associated mortality. NTM are naturally resistant to a variety of antibiotics, complicating treatment. We conducted a literature assessment on the efficacy of bedaquiline in treating NTM species in vitro and in vivo (animal models and humans); meta-analyses were performed where possible.

Bedaquiline for treatment of non-tuberculous mycobacteria (NTM): a systematic review and meta-analysis.

Coinfection with two phylogenetically distinct Human Immunodeficiency Virus-1 (HIV-1) variants might provide an opportunity for rapid viral expansion and the emergence of fit variants that drive disease progression. However, autologous neutralising immune responses are known to drive Envelope (Env) diversity which can either enhance replicative capacity, have no effect, or reduce viral fitness. This study investigated whether in vivo outgrowth of coinfecting variants was linked to pseudovirus and infectious molecular clones' infectivity to determine whether diversification resulted in more fit virus with the potential to increase disease progression.

The evolution of envelope function during coinfection with phylogenetically distinct human immunodeficiency virus.

Phenotypic drug susceptibility testing (pDST) remains the gold standard for  complex drug resistance determination. Next generation sequencing technologies can identify heteroresistant populations at low frequencies, but little is known about the impact of heteroresistance on bedaquiline (BDQ) pDST results. We simulated heteroresistance using  generated  mutants mixed with the progenitor strain at various percentages (1-20%) and did pDST using MGIT960 culture (1 and 2 μg/mL BDQ concentrations). Targeted Next Generation Sequencing (tNGS) was used to quantify the mutant sub-population in growth control tubes, which were expected to maintain the mutant: wild type proportion throughout the assay. Growth units of these growth control tubes were also comparable with minor differences in time-to-positivity between ratio mixtures. Only when intermediate results were considered could BDQ heteroresistance be detected at frequencies of approximately 1% by pDST at a critical concentration of 1 μg/mL using BACTEC MGIT960 coupled with EpiCenter TBeXiST software. The ability of pDST, a widely available DST technique, to reveal the presence of BDQ-resistant subpopulations at the phenotypic testing stage could improve resistance determination and potentially reduce time to effective treatment.

Unpacking bedaquiline hetero-resistance: the importance of intermediate profiles for phenotypic drug-susceptibility testing.

Phenotypic drug susceptibility testing (pDST) remains a widely used standard for determination of resistance for several drugs for the  complex. Next-generation sequencing technologies can identify heteroresistant populations at low frequencies, but little is known about the impact of heteroresistance on bedaquiline (BDQ) pDST results. We simulated heteroresistance using -generated  mutants mixed with the progenitor strain at various percentages (1%-20%) and performed pDST using the BACTEC MGIT 960 platform (1 and 2 µg/mL BDQ concentrations) coupled with EpiCenter TB-eXtended individual drug Susceptibility Testing software. Targeted next-generation sequencing was used to quantify the mutant subpopulation in growth control tubes, which were expected to maintain the mutant: wild-type proportion throughout the assay. Growth units of these growth control tubes were also comparable with minor differences in time to positivity between ratio mixtures. Only when intermediate results were considered (i.e., when growth units in a drug-containing tube reach the threshold for resistance but only after a further week of incubation) could BDQ heteroresistance be detected at frequencies of approximately 1% by pDST at a critical concentration of 1 µg/mL. These intermediate results, commonly disregarded during routine testing, could lead to earlier detection of BDQ resistance and may avert adverse clinical outcomes. The ability of pDST, a widely available DST technique, to reveal the presence of BDQ-resistant subpopulations at the phenotypic testing stage could improve resistance determination and potentially reduce time to effective treatment.

Unpacking bedaquiline heteroresistance: the importance of intermediate profiles for phenotypic drug susceptibility testing.

Whole-genome sequencing (WGS) holds promise for accurate and comprehensive diagnosis of drug resistance in  and identification of transmission events. 'Early positive cultures' (EPC) are increasingly used when WGS is implemented to guide clinical care to reduce the turnaround time. We performed a systematic literature review to compare methods used for EPC-based WGS and performed an individual sample data meta-analysis to identify variables associated with bioinformatic quality measures. Of 423 studies identified, 15 met eligibility criteria. We analysed 1,065 FASTQ files from 11 studies using Illumina sequencing; 96.1% passed all quality control thresholds. Median genome coverage was 65× (IQR, 63-82), with a pooled mapping percentage of 91.2%. The meta-analysis showed that the number of sequencing cycles was significantly associated with improved sequencing quality, while other laboratory variables had no consistent effect. Based on these findings, we suggest replacing the term EPC with 'clinical primary culture' and propose a standardized workflow and reporting checklist for WGS on primary Mycobacteria Growth Indicator Tube (MGIT) cultures.

Article	Year
Bedaquiline for treatment of non-tuberculous mycobacteria (NTM): a systematic review and meta-analysis. The Journal of antimicrobial chemotherapy\| PubMed ID: 38134888	2024
The evolution of envelope function during coinfection with phylogenetically distinct human immunodeficiency virus. BMC infectious diseases\| PubMed ID: 39251948	2024
Unpacking bedaquiline hetero-resistance: the importance of intermediate profiles for phenotypic drug-susceptibility testing. bioRxiv : the preprint server for biology\| PubMed ID: 40161652	2025
Unpacking bedaquiline heteroresistance: the importance of intermediate profiles for phenotypic drug susceptibility testing. Antimicrobial agents and chemotherapy\| PubMed ID: 40689761	2025
cultured in MGIT media for whole-genome sequencing application: a systematic literature review and meta-analysis. Microbial genomics\| PubMed ID: 41277813	2025

Shatha Omar

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