Biology subjects

van Ingen, J.

Publications and source records attributed to van Ingen, J..

4 recordsLinked to original sources

bioRxiv2026

Analytical concordance of targeted next-generation sequencing and whole-genome sequencing for Mycobacterium tuberculosis drug resistance and lineage determination in West Java, Indonesia

As drug-resistant tuberculosis (DR-TB) cases rise, resistance detection in a timely manner is essential to lead effective treatment and limit transmission. Targeted next-generation sequencing (tNGS) offers quick results with multiple important drugs covered, but assessments regarding its performance for DR-TB diagnostic use compared to whole genome sequencing (WGS) as the most comprehensive genomic-based tool are still limited. This cross-sectional study compared resistance profiles generated by Deeplex Myc-TB tNGS assay with WGS for 116 prospectively-collected rifampicin resistant TB samples from West Java, Indonesia. All 116 samples were subject to paired analysis, the clinical samples were split to be directly processed for tNGS and to be cultivated for culture-based WGS. Both WGS and tNGS were carried out using Illumina MiSeq platform. High concordance of tNGS and WGS were observed across thirteen anti-TB drugs evaluated, particularly for drugs included in the BPaLM regimen. Isoniazid had the lowest concordance of 86.73%. Of 116 samples, 31.03% (n = 36) had discrepant resistance calling from the two methods for one or more drugs, which came from 73 discordant variants identification. The most common source of discrepancy was when tNGS detected a resistance-conferring mutation while WGS did not (54.8%). tNGS could detect mixed infection better than WGS, but WGS was superior in identifying detailed major Mycobacterium tuberculosis lineage of the sample. tNGS showed a good level concordance with WGS in detecting resistance-conferring mutations in rifampicin-resistant TB samples, with a more rapid turnaround time. Continuous update to tNGS panel and mutation catalogue is needed to keep the tool clinically relevant. ImportanceDrug-resistant tuberculosis (DR-TB) continues to pose worldwide threat, and newer diagnostic tools to generate quick, comprehensive resistance profile are crucial to provide timely appropriate treatment. Targeted next-generation sequencing (tNGS) is a promising new alternative, but more evidence on its performance is needed to support programmatic adoption. By analysing DR-TB samples with both tNGS and whole genome sequencing (WGS) and evaluating their results agreement, this study shows that tNGS works just as well as WGS in detecting TB drug resistance-conferring mutations, confirming its potential for routine diagnostic use. This study also observed that while WGS is superior in identifying Mycobacterium tuberculosis lineage with high resolution, it did not detect mixed infection better than tNGS. Notably, this study demonstrated that tNGS is clinically relevant for DR-TB detection in a high burden setting, providing evidence for programmatic consideration in Indonesia and other settings with similar demographics and TB situation.

genomics↗

bioRxiv2026

Activity of a three-phage combination against Mycobacterium tuberculosis in disease-relevant conditions

Phage therapy offers promise to combat antimicrobial resistance, including drug-resistant tuberculosis (TB). Understanding phage activity against Mycobacterium tuberculosis (Mtb) adapted to physiologic microenvironments, such as hypoxia and acidity in granulomas, is essential since these conditions induce non-replicating states. We evaluated a phage combination against Mtb under hypoxic, acidic (pH 5.5), and stationary-phase conditions in vitro. In planktonic Mtb growth conditions, phage concentrations increased around day seven followed by a significant reduction in Mtb H37Rv load, which was maintained over 31 days. Phage addition prevented regrowth was observed with rifampicin and isoniazid alone. Individual phage stability was differentially affected by acidic media conditions, resulting in variability of antimycobacterial activity. In hypoxic conditions and stationary growth experiments, phage titers remained stable over time with no change in mycobacterial load compared to controls. Model-based predictions were able to adequately capture phage-mycobacterial interactions with and without rifampicin. The lack of antimycobacterial activity in assays with non-replicating mycobacteria suggest that phages need actively replicating mycobacteria to exert lytic activity. Stable phage concentrations in assays with non-replicating mycobacteria suggests low grade phage replication in these conditions. Established models can support future study design through simulations of different experimental scenarios.

microbiology↗

bioRxiv2026

A new stress-response pathway in Mycobacterium tuberculosis

Metabolic adaptations are key in the virulence of the pathogen Mycobacterium tuberculosis (Mtb). However, our current understanding of these adaptations is limited to common pathways in central carbon metabolism. Here we apply an untargeted bottom-up approach to discover unknown stress-responsive metabolites and their biosynthetic enzyme. We show that upon exposure to hypoxia, nitric oxide and activated macrophages, Mtb rapidly produces high levels of an unknown metabolite that we identify as 6-{gamma}-amino-butyric acid-trehalose (GABA-trehalose). Formation of millimolar GABA-trehalose levels under these stresses is driven by a rapid rise in GABA, which is also excreted. We demonstrate that GABA-trehalose is produced from GABA and trehalose by the uncharacterized ATP-grasp enzyme Rv1722, involving a carboxylate-hydroxyl ligation that is non-canonical for ATP-grasp enzymes. Phylogenetic analyses demonstrate that the gene rv1722 is present in most slow-growing mycobacteria but absent in most rapid-growing mycobacteria. While the role of GABA-trehalose in Mtb metabolism remains unclear, we postulate that the increased NADH/NAD+ ratio under hypoxia and nitric oxide exposure promotes GABA formation and inhibits its breakdown, leading to GABA accumulation and excretion. Rv1722-driven coupling of GABA and trehalose constitutes an alternative to excretion that conserves carbon and nitrogen. Taken together, our bottom-up approach reveals a new stress-response pathway in Mtb that rapidly produces large quantities of GABA-trehalose. These findings extend our knowledge of the metabolic adaptations that a major human pathogen utilizes in response to immune system-imposed stresses.

biochemistry↗

bioRxiv2021

An optimized protocol for dual RNA-Seq of human macrophages infected with Mycobacterium avium

Recently, dual RNA-sequencing (RNA-Seq) has been identified as a powerful tool to study host-pathogen interactions, which is particularly interesting for intracellular pathogens such as mycobacteria. However, due to the complexity of obtaining human host cells, many models rely on the usage of host cells derived from animals or cell lines, which does not accurately mimic the situation in the patient. Furthermore, due to the severe disbalance in host and pathogen RNA content, it is difficult to obtain sufficient sequencing depth for the infecting pathogen. Here, we present an optimized method to perform dual RNA-sequencing on human monocyte-derived macrophages (hMDMs) infected with Mycobacterium avium (M. avium). It is likely that, with slight modifications in multiplicity of infection (MOI) to account for differences in virulence, this methodology will be applicable for other difficult-to-lyse intracellular mycobacteria.

genomics↗