Iron-sulphur (Fe-S) clusters are ubiquitous co-factors which require multi-protein systems for their synthesis. In Mycobacterium tuberculosis, the Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466 operon ...(suf operon) encodes the primary Fe-S cluster biogenesis system. The first gene in this operon, Rv1460, shares homology with the cyanobacterial SufR, which functions as a transcriptional repressor of the sufBCDS operon. Rv1460's function in M. tuberculosis has however not been determined. In this study, we demonstrate that M. tuberculosis mutants lacking a functional Rv1460 protein are impaired for growth under standard culture conditions. Elevated expression of Rv1460 and Rv1461 was observed in the mutant, implicating Rv1460 in the regulation of the suf operon. Binding of an Fe-S cluster to purified recombinant Rv1460 was confirmed by UV-visible spectroscopy and circular dichroism. Furthermore, three conserved cysteine residues, C203, C216 and C244, proposed to provide ligands for the coordination of an Fe-S cluster, were shown to be required for the function of Rv1460 in M. tuberculosis. Rv1460 therefore seems to be functionally analogous to cyanobacterial SufR.
Whole genome sequencing (WGS) holds great potential for the management and control of tuberculosis. Accurate analysis of samples with low mycobacterial burden, which are characterized by low (40%) ...levels of contamination, is challenging. We created the MAGMA (Maximum Accessible Genome for Mtb Analysis) bioinformatics pipeline for analysis of clinical Mtb samples. High accuracy variant calling is achieved by using a long seedlength during read mapping to filter out contaminants, variant quality score recalibration with machine learning to identify genuine genomic variants, and joint variant calling for low Mtb coverage genomes. MAGMA automatically generates a standardized and comprehensive output of drug resistance information and resistance classification based on the WHO catalogue of Mtb mutations. MAGMA automatically generates phylogenetic trees with drug resistance annotations and trees that visualize the presence of clusters. Drug resistance and phylogeny outputs from sequencing data of 79 primary liquid cultures were compared between the MAGMA and MTBseq pipelines. The MTBseq pipeline reported only a proportion of the variants in candidate drug resistance genes that were reported by MAGMA. Notable differences were in structural variants, variants in highly conserved rrs and rrl genes, and variants in candidate resistance genes for bedaquiline, clofazmine, and delamanid. Phylogeny results were similar between pipelines but only MAGMA visualized clusters. The MAGMA pipeline could facilitate the integration of WGS into clinical care as it generates clinically relevant data on drug resistance and phylogeny in an automated, standardized, and reproducible manner.
Abstract Animal tuberculosis significantly challenges global health, agriculture, and wildlife conservation efforts. Mycobacterial cultures are resource-intensive, time-consuming, and challenged by ...heterogeneous populations. In this study, we employed a culture-independent approach, using targeted long-read-based next-generation sequencing (tNGS), to investigate the mycobacterial composition in 60 DNA samples extracted from Mycobacterium bovis infected culture-confirmed African buffalo tissue. We detected mycobacterial DNA in 93.3% of the samples and the sensitivity for detecting Mycobacterium tuberculosis complex (MTBC) was 91.7%, demonstrating a high concordance of our culture-independent tNGS approach with mycobacterial culture results. In five samples, we identified heterogenous mycobacterial populations with various non-tuberculous mycobacteria, including members of the Mycobacterium avium complex (MAC), M. smegmatis , and M. komaniense . The latter Mycobacterium species was described in South Africa from bovine nasal swabs and environmental samples from the Hluhluwe-iMfolozi Park, which was the origin of the buffalo samples in the present study. This finding suggests that exposure to environmental mycobacteria may confound detection of MTBC in wildlife. In conclusion, our approach represents a promising alternative to conventional methods for detecting mycobacterial DNA. This high-throughput technique enables rapid differentiation of heterogeneous mycobacterial populations, which will contribute valuable insights into the epidemiology, pathogenesis, and microbial synergy during mycobacterial infections.
Molecular detection of bedaquiline resistant tuberculosis is challenging as only a small proportion of mutations in candidate bedaquiline resistance genes have been statistically associated with ...phenotypic resistance. We introduced two mutations, atpE Ile66Val and Rv0678 Thr33Ala, in the Mycobacterium tuberculosis H37Rv reference strain using homologous recombineering or recombination to investigate the phenotypic effect of these mutations. The genotype of the resulting strains was confirmed by Sanger- and whole genome sequencing, and bedaquiline susceptibility was assessed by minimal inhibitory concentration (MIC) assays. The impact of the mutations on protein stability and interactions was predicted using mutation Cutoff Scanning Matrix (mCSM) tools. The atpE Ile66Val mutation did not elevate the MIC above the critical concentration (MIC 0.25-0.5 µg/ml), while the MIC of the Rv0678 Thr33Ala mutant strains (> 1.0 µg/ml) classifies the strain as resistant, confirming clinical findings. In silico analyses confirmed that the atpE Ile66Val mutation minimally disrupts the bedaquiline-ATP synthase interaction, while the Rv0678 Thr33Ala mutation substantially affects the DNA binding affinity of the MmpR transcriptional repressor. Based on a combination of wet-lab and computational methods, our results suggest that the Rv0678 Thr33Ala mutation confers resistance to BDQ, while the atpE Ile66Val mutation does not, but definite proof can only be provided by complementation studies given the presence of secondary mutations.
The next-generation, short-read sequencing technologies that generate comprehensive, whole-genome data with single nucleotide resolution have already advanced tuberculosis diagnosis, treatment, ...surveillance, and source investigation. Their high costs, tedious and lengthy processes, and large equipment remain major hurdles for research use in high tuberculosis burden countries and implementation into routine care. The portable next-generation sequencing devices developed by Oxford Nanopore Technologies (ONT) are attractive alternatives due to their long-read sequence capability, compact low-cost hardware, and continued improvements in accuracy and throughput. A systematic review of the published literature demonstrated limited uptake of ONT sequencing in tuberculosis research and clinical care. Of the 12 eligible articles presenting ONT sequencing data on at least one Mycobacterium tuberculosis sample, four addressed software development for long-read ONT sequencing data with potential applications for M. tuberculosis. Only eight studies presented results of ONT sequencing of M. tuberculosis, of which five performed whole-genome and three did targeted sequencing. Based on these findings, we summarize the standard processes, reflect on the current limitations of ONT sequencing technology, and the research needed to overcome the main hurdles. The low capital cost, portable nature and continued improvement in the performance of ONT sequencing make it an attractive option for sequencing for research and clinical care, but limited data are available on its application in the tuberculosis field. Important research investment is needed to unleash the full potential of ONT sequencing for tuberculosis research and care.
Heteroresistant Mycobacterium tuberculosis infections (defined as concomitant infection with drug-resistant and drug-susceptible strains) may explain the higher risk of poor tuberculosis treatment ...outcomes observed among patients with mixed-strain M. tuberculosis infections. We investigated the clinical effect of mixed-strain infections while controlling for pretreatment heteroresistance in a population-based sample of patients with tuberculosis starting first-line tuberculosis therapy in Botswana.
We performed 24-locus mycobacterial interspersed repetitive unit-variable number tandem-repeat analysis and targeted deep sequencing on baseline primary cultured isolates to detect mixed infections and heteroresistance, respectively. Drug-sensitive, micro-heteroresistant, macro-heteroresistant, and fixed-resistant infections were defined as infections in which the frequency of resistance was <0.1%, 0.1%-4%, 5%-94%, and ≥95%, respectively, in resistance-conferring domains of the inhA promoter, the katG gene, and the rpoB gene.
Of the 260 patients with tuberculosis included in the study, 25 (9.6%) had mixed infections and 30 (11.5%) had poor treatment outcomes. Micro-heteroresistance, macro-heteroresistance, and fixed resistance were found among 11 (4.2%), 2 (0.8%), and 11 (4.2%), respectively, for isoniazid and 21 (8.1%), 0 (0%), and 10 (3.8%), respectively, for rifampicin. In multivariable analysis, mixed infections but not heteroresistant infections independently predicted poor treatment outcomes.
Among patients starting first-line tuberculosis therapy in Botswana, mixed infections were associated with poor tuberculosis treatment outcomes, independent of heteroresistance.
The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient ...care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs.
To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance.
We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool.
The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci.
These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs.
Tuberculosis lymphadenitis (TBL) is the most common extrapulmonary tuberculosis (EPTB) manifestation. Xpert MTB/RIF Ultra (Ultra) is a World Health Organization-endorsed diagnostic test, but ...performance data for TBL, including on noninvasive specimens, are limited. Fine-needle aspiration biopsy specimens (FNABs) from outpatients (≥18 years) with presumptive TBL (
= 135) underwent (i) routine Xpert MTB/RIF testing (later with Ultra once programmatically available), (ii) MGIT 960 culture (if Xpert or Ultra negative or rifampicin resistant), and (iii) study Ultra testing. Concentrated paired urine specimens underwent Ultra testing. Primary analyses used a microbiological reference standard (MRS). In a head-to-head comparison (
= 92) of an FNAB study Ultra and Xpert, Ultra had increased sensitivity (91% 95% confidence interval: 79, 98 versus 72% 57, 84;
= 0.016) and decreased specificity (76% 61, 87 versus 93% 82, 99;
= 0.020) and diagnosed patients not on treatment. Neither HIV nor alternative reference standards affected sensitivity and specificity. In patients with both routine and study Ultra tests, the latter detected more cases (+20% 0, 42;
= 0.034), and false-negative study Ultra results were more inhibited than true-positive results. Study Ultra false positives had less mycobacterial DNA than true positives (trace-positive proportions, 59% 13/22 versus 12% 5/51;
< 0.001). "Trace" exclusion or recategorization removed potential benefits offered over Xpert. Urine Ultra tests had low sensitivity (18% 7, 35). Ultra testing on FNABs is highly sensitive and detects more TBL than Xpert (Ultra still missed some cases due in part to inhibition). Patients with FNAB Ultra-positive "trace" results, most of whom will be culture negative, may require additional clinical investigation. Urine Ultra testing could reduce the number of patients needing invasive sampling.
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