matrix-based-dna-extraction-for-targeted-next-generation-sequencing

Matrix-based DNA Extraction for Targeted Next-Generation Sequencing on Decontaminated Sputum Samples

Next-generation sequencing (NGS) is now recognized as a powerful tool for timely and accurate drug-resistant tuberculosis (DR-TB) diagnosis. Targeted NGS ...

mycobacterial-dna-extraction-using-bead-beating-custom-buffer

Mycobacterial DNA Extraction using Bead Beating in Custom Buffer Followed by NGS Workflow

Tuberculosis is a deadly disease, and the emergence of antibiotic drug resistance in the causative agent bacterium, Mycobacterium tuberculosis, worsens ...

amplicon-sequencing-using-the-long-read-sequencing-technologies

Amplicon Sequencing using the Long-Read Sequencing Technologies

The World Health Organization (WHO) continues to emphasize the urgent need for a rapid, cost-effective, and user-friendly diagnostic method for ...

Stellenbosch University

Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations.

Rifampicin reduces susceptibility to ofloxacin in rifampicin-resistant Mycobacterium tuberculosis through efflux.

The shortcomings of conventional tuberculosis treatments resulting from the development of drug resistance in Mycobacterium tuberculosis drive a need for the formulation of novel therapeutic agents. The diarylquinoline class of drugs such as bedaquiline was recently approved for the treatment of multidrug-resistant strains of tuberculosis, primarily targeting c and ε subunits of the ATP synthases. Yet resistance to bedaquiline has already been reported. Therefore, Rv1311 was used as the target for the identification of possible inhibitors against the M. tuberculosis. The structure of Rv1311 was predicted and common feature pharmacophore models were generated which facilitated the identification of potential inhibitors in the ZINC database. The activities of the selected molecules were compared with known inhibitors of the ATP synthase using quantitative structure-activity relationship. The ZINC classified inhibitors showed comparable predicted activities with that of known inhibitors. Furthermore, the inhibitory behavior of the studied drug molecules was experimentally determined using in vitro techniques and showed the minimum inhibitory concentration as low as 25 μM. The resulted outcomes provide a deeper insight into the structural basis of Rv1311 inhibitions and can facilitate the process of drug design against tuberculosis.

Structure based identification of novel inhibitors against ATP synthase of Mycobacterium tuberculosis: A combined in silico and in vitro study.

Bedaquiline Microheteroresistance after Cessation of Tuberculosis Treatment.

Rifampicin was discovered in 1965 and remains one of the most important drugs in tuberculosis treatment that is valued for its sterilizing activity and ability to shorten treatment. Antimicrobial activity of rifampicin was initially proved in vitro; subsequently numerous in vivo studies showed the bactericidal properties and dose-dependent effect of rifampicin. Rifampicin was first during the late 1960s to treat patients suffering from chronic drug-resistant pulmonary TB. Decades later, rifampicin continues to be studied with particular emphasis on whether higher doses could shorten the duration of treatment without increasing relapse or having adverse effects. Lesion-specific drug penetration and pharmacokinetics of rifampicin are improving our understanding of effective concentration while potentially refining drug regimen designs. Another prospective aspect of high-dose rifampicin is its potential use in treating discrepant mutation thereby eliminating the need for MDR treatment. To date, several clinical trials have shown the safety, efficacy, and tolerability of high-dose rifampicin. Currently, high-dose rifampicin has been used successfully in a routine clinical setting for the treatment of high-risk patients. However, the WHO and other relevant policy makers have not committed to implementing a controlled rollout thereof. This review describes the course that rifampicin has travelled to the present-day exploration of high-dose rifampicin treatment.

Evolution of rifampicin treatment for tuberculosis.

Nicotinamide-nucleotide adenylyl transferase (Rv2421c) was selected as a potential drug target, because it has been shown, in vitro, to be essential for Mycobacterium tuberculosis growth. It is conserved between mycobacterium species, is up-regulated during dormancy, has a known 3D crystal structure and has no known human homologs. A model of Rv2421c in complex with nicotinic acid adenine dinucleotide and magnesium ion was constructed and subject tovirtual ligand screening against the Prestwick Chemical Library and the ZINC database, which yielded 155 potential hit molecules. Of the 155 compounds identified five were pursued further using an IC50 based 3D-QSAR study. The 3D-QSAR model validated the inhibition properties of the five compounds based on R2 value of 0.895 and Q2 value of 0.944 compared to known inhibitors of Rv2421c. Higher binding affinities was observed for the novel ZINC13544129 and two FDA approved compounds (Novobiocin sodium salt, Sulfasalazine). Similarly, the total interaction energy was found to be the highest for Cromolyn disodium system (-418.88 kJ/mol) followed by Novobiocin (-379.19 kJ/mol) and Sulfasalazine with (-330.13 kJ/mol) compared to substrate DND having (-185.52 kJ/mol). Subsequent in vitro testing of the five compounds identified Novobiocin sodium salt with activity against Mycobacterium tuberculosis at 50 μM, 25μM and weakly at 10μM concentrations. Novobiocin salt interacts with a MG ion and active site residues His20, Thr86, Gly107 and Leu164 similar to substrate DND of Mycobacterium tuberculosis Rv2421c. Additional in silico structural analysis of known Novobiocin sodium salt derivatives against Rv2421c suggest Coumermycin as a promising alternative for the treatment of Mycobacterium tuberculosis based on large number of hydrogen bond interactions with Rv2421c similar in comparison to Novobiocin salt and substrate DND.

In silico repurposing of a Novobiocin derivative for activity against latency associated Mycobacterium tuberculosis drug target nicotinate-nucleotide adenylyl transferase (Rv2421c).

Whole genome sequencing (WGS) can investigate the entire Mycobacterium tuberculosis (Mtb) genome but currently requires large amounts of mycobacterial DNA, necessitating culture. Culture-free Mtb WGS could revolutionize the clinical use of WGS but is hampered by the high viscosity, low mycobacterial load, and high contamination with bacterial and human DNA in sputum samples. To improve the sputum liquefaction and decontamination step prior to DNA extraction, we assessed the efficiency of Myco-TB, MycoPrep, and Sputolysin with/without TiKa-Kic in liquefying and decontaminating sputum and aimed to evaluate the effect of these approaches on mycobacterial viability, and Mtb DNA quality and quantity. Experiments using spiked sputum samples showed that Myco-TB and BD MycoPrep with standard (15 min) or increased (30 min) incubation time, but not reduced (7,5 min) incubation time performed well in liquefying and decontaminating sputum. No difference in DNA quality or quantity, contamination, or the amount of human DNA present was observed. In comparison, Sputolysin with/without TiKa-Kic was less effective for liquefaction and decontamination of sputum. PCR amplification of the human GAPDH gene after sputum treatment, showed the presence of human DNA in all samples, regardless of sputum treatment. Focused efforts are needed to deplete contaminating DNA for culture-free Mtb WGS.

Optimizing liquefaction and decontamination of sputum for DNA extraction from Mycobacterium tuberculosis.

Mycobacterium tuberculosis whole-genome sequencing (WGS) has been widely used for genotypic drug susceptibility testing (DST) and outbreak investigation. For both applications, Illumina technology is used by most public health laboratories; however, Nanopore technology developed by Oxford Nanopore Technologies has not been thoroughly evaluated. The aim of this study was to determine whether Nanopore sequencing data can provide equivalent information to Illumina for transmission clustering and genotypic DST for M tuberculosis.

Evaluation of Nanopore sequencing for Mycobacterium tuberculosis drug susceptibility testing and outbreak investigation: a genomic analysis.

Mycobacterium tuberculosis whole-genome sequencing (WGS) is a powerful tool as it can provide data on population diversity, drug resistance, disease transmission, and mixed infections. Successful WGS is still reliant on high concentrations of DNA obtained through M. tuberculosis culture. Microfluidics technology plays a valuable role in single-cell research but has not yet been assessed as a bacterial enrichment strategy for culture-free WGS of M. tuberculosis. In a proof-of-principle study, we evaluated the use of Capture-XT, a microfluidic lab-on-chip cleanup and pathogen concentration platform to enrich M. tuberculosis bacilli from clinical sputum specimens for downstream DNA extraction and WGS. Three of the four (75%) samples processed by the microfluidics application passed the library preparation quality control, compared to only one of the four (25%) samples not enriched by the microfluidics M. tuberculosis capture application. WGS data were of sufficient quality, with mapping depth of ≥25× and 9 to 27% of reads mapping to the reference genome. These results suggest that microfluidics-based M. tuberculosis cell capture might be a promising method for M. tuberculosis enrichment in clinical sputum samples, which could facilitate culture-free M. tuberculosis WGS.  Diagnosis of tuberculosis is effective using molecular methods; however, a comprehensive characterization of the resistance profile of Mycobacterium tuberculosis often requires culturing and phenotypic drug susceptibility testing or culturing followed by whole-genome sequencing (WGS). The phenotypic route can take anywhere from 1 to >3 months to result, by which point the patient may have acquired additional drug resistance. The WGS route is a very attractive option; however, culturing is the rate-limiting step. In this original article, we provide proof-of-principle evidence that microfluidics-based cell capture can be used on high-bacillary-load clinical samples for culture-free WGS.

Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing.

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.

Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA.

Bacteria adapt to changes in their natural environment through a network of stress responses that enable them to alter their gene expression to survive in the presence of stressors, including antibiotics. These stress responses can be specific to the type of stress and the general stress response can be induced in parallel as a backup mechanism. In Gram-negative bacteria, various envelope stress responses are induced upon exposure to antibiotics that cause damage to the cell envelope or result in accumulation of toxic metabolic by-products, while the heat shock response is induced by antibiotics that cause misfolding or accumulation of protein aggregates. Antibiotics that result in the production of reactive oxygen species (ROS) induce the oxidative stress response and those that cause DNA damage, directly and through ROS production, induce the SOS response. These responses regulate the expression of various proteins that work to repair the damage that has been caused by antibiotic exposure. They can contribute to antibiotic resistance by refolding, degrading or removing misfolded proteins and other toxic metabolic by-products, including removal of the antibiotics themselves, or by mutagenic DNA repair. This review summarizes the stress responses induced by exposure to various antibiotics, highlighting their interconnected nature, as well the roles they play in antibiotic resistance, most commonly through the upregulation of efflux pumps. This can be useful for future investigations targeting these responses to combat antibiotic-resistant Gram-negative bacterial infections.

ArticleTotal : 3	Year
Publication title	2025
Publication title	2025
Publication title	2025

Article	Year
Rifampicin reduces susceptibility to ofloxacin in rifampicin-resistant Mycobacterium tuberculosis through efflux. American journal of respiratory and critical care medicine\| PubMed ID: 21512166	2011
Structure based identification of novel inhibitors against ATP synthase of Mycobacterium tuberculosis: A combined in silico and in vitro study. International journal of biological macromolecules\| PubMed ID: 31121229	2019
Bedaquiline Microheteroresistance after Cessation of Tuberculosis Treatment. The New England journal of medicine\| PubMed ID: 31141643	2019
Evolution of rifampicin treatment for tuberculosis. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases\| PubMed ID: 31247337	2019
In silico repurposing of a Novobiocin derivative for activity against latency associated Mycobacterium tuberculosis drug target nicotinate-nucleotide adenylyl transferase (Rv2421c). PloS one\| PubMed ID: 34727137	2021
Optimizing liquefaction and decontamination of sputum for DNA extraction from Mycobacterium tuberculosis. Tuberculosis (Edinburgh, Scotland)\| PubMed ID: 34906896	2022
Evaluation of Nanopore sequencing for Mycobacterium tuberculosis drug susceptibility testing and outbreak investigation: a genomic analysis. The Lancet. Microbe\| PubMed ID: 36549315	2023
Microfluidic Capture of Mycobacterium tuberculosis from Clinical Samples for Culture-Free Whole-Genome Sequencing. Microbiology spectrum\| PubMed ID: 37358439	2023
Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA. Scientific reports\| PubMed ID: 38689002	2024
Antibiotic-induced stress responses in Gram-negative bacteria and their role in antibiotic resistance. The Journal of antimicrobial chemotherapy\| PubMed ID: 40053699	2025

Melanie Grobbelaar

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