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The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs, now modified to include detection of Delta (B.1.617.2) and Omicron (B.1.1.529) sub-variants. The SMB-VOC assay targets the signature codons 501, 484 and 452 in the SARS-CoV-2 spike protein which we show can specifically detect and differentiate all known VOCs including the Omicron subvariants (BA.1, BA.2, BA.2.12.1, BA.4/BA.5). The limit of detection (LOD) of the assay was 20, 22 and 36 genomic equivalents (GE) per reaction with the Delta, Omicron BA.1 and BA.2 respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% (81/86) of the specimens as WT or VOCs and 6% (5/86) of the tests producing indeterminate results compared to sequencing. Sanger sequencing also failed for four samples. None of the specimens were incorrectly identified as WT or as a different VOC by our assay. Thus, excluding specimens with indeterminant results, the assay was 100% sensitive and 100% specific compared to Sanger sequencing for variant identification. This new assay concept can be easily expanded to add newer variants and can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance. [ABSTRACT FROM AUTHOR]

H37Rv is the most widely used Mycobacterium tuberculosis strain, and its genome is globally used as the M. tuberculosis reference sequence. Here, we present Bact-Builder, a pipeline that uses consensus building to generate complete and accurate bacterial genome sequences and apply it to three independently cultured and sequenced H37Rv aliquots of a single laboratory stock. Two of the 4,417,942 base-pair long H37Rv assemblies are 100% identical, with the third differing by a single nucleotide. Compared to the existing H37Rv reference, the new sequence contains ~6.4 kb additional base pairs, encoding ten new regions that include insertions in PE/PPE genes and new paralogs of esxN and esxJ, which are differentially expressed compared to the reference genes. New sequencing and de novo assemblies with Bact-Builder confirm that all 10 regions, plus small additional polymorphisms, are also present in the commonly used H37Rv strains NR123, TMC102, and H37Rv1998. Thus, Bact-Builder shows promise as an improved method to perform accurate and reproducible de novo assemblies of bacterial genomes, and our work provides important updates to the primary M. tuberculosis reference genome. H37Rv is the most widely used Mycobacterium tuberculosis strain, and its genome is the reference sequence for this pathogen. Here, Chitale et al. present a bioinformatic pipeline for accurate assembly of bacterial genome sequences, and use it to provide important updates to the M. tuberculosis reference genome. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis can adapt to changing environments by non-heritable mechanisms. Frame-shifting insertions and deletions (indels) may also participate in adaptation through gene disruption, which could be reversed by secondary introduction of a frame-restoring indel. We present ScarTrek, a program that scans genomic data for indels, including those that together disrupt and restore a gene's reading frame, producing "frame-shift scars" suggestive of reversible gene inactivation. We use ScarTrek to analyze 5977 clinical M. tuberculosis isolates. We show that indel frequency inversely correlates with genomic linguistic complexity and varies with gene-position and gene-essentiality. Using ScarTrek, we detect 74 unique frame-shift scars in 48 genes, with a 3.74% population-level incidence of unique scar events. We find multiple scars in the ESX-1 gene cluster. Six scars show evidence of convergent evolution while the rest shared a common ancestor. Our results suggest that sequential indels are a mechanism for reversible gene silencing and adaptation in M. tuberculosis. Bacterial adaptation through frame-shifting insertions and deletions (indels) could be reversed by secondary introduction of a frame-restoring indel. Here, the authors develop ScarTrek, a program that scans genomic data for different indels, and analyze 5977 clinical M. tuberculosis isolates for indel frequency. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Background: There is a growing interest in the use of F-18 FDG PET-CT to monitor tuberculosis (TB) treatment response. Tuberculosis lung lesions are often complex and diffuse, with dynamic changes during treatment and persisting metabolic activity after apparent clinical cure. This poses a challenge in quantifying scan-based markers of burden of disease and disease activity. We used semi-automated, whole lung quantification of lung lesions to analyse serial FDG PET-CT scans from the Catalysis TB Treatment Response Cohort to identify characteristics that best correlated with clinical and microbiological outcomes. Results: Quantified scan metrics were already associated with clinical outcomes at diagnosis and 1 month after treatment, with further improved accuracy to differentiate clinical outcomes after standard treatment duration (month 6). A high cavity volume showed the strongest association with a risk of treatment failure (AUC 0.81 to predict failure at diagnosis), while a suboptimal reduction of the total glycolytic activity in lung lesions during treatment had the strongest association with recurrent disease (AUC 0.8 to predict pooled unfavourable outcomes). During the first year after TB treatment lesion burden reduced; but for many patients, there were continued dynamic changes of individual lesions. Conclusions: Quantification of FDG PET-CT images better characterised TB treatment outcomes than qualitative scan patterns and robustly measured the burden of disease. In future, validated metrics may be used to stratify patients and help evaluate the effectiveness of TB treatment modalities. [ABSTRACT FROM AUTHOR]

Correction to: I Nature Communications i https://doi.org/10.1038/s41467-022-34853-x, published online 18 November 2022 The original version of the Supplementary Information associated with this Article was missing a list of Supplementary References. Supplementary information Graph: Updated Supplementary Information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-022-35402-2. [Extracted from the article]

To study the evolution of drug resistance, we genetically and biochemically characterized Mycobacterium tuberculosis strains selected in vitro for ethambutol resistance. Mutations in decaprenylphosphoryl-β-D-arabinose (DPA) biosynthetic and utilization pathway genes Rv3806c, Rv3792, embB and embC accumulated to produce a wide range of ethambutol minimal inhibitory concentrations (MICs) that depended on mutation type and number. Rv3806c mutations increased DPA synthesis, causing MICs to double from 2 to 4 μg/ml in a wild-type background and to increase from 16 to 32 μg/ml in an embB codon 306 mutant background. Synonymous mutations in Rv3792 increased the expression of downstream embC, an ethambutol target, resulting in MICs of 8 μg/ml. Multistep selection was required for high-level resistance. Mutations in embC or very high embC expression were observed at the highest resistance level. In clinical isolates, Rv3806c mutations were associated with high-level resistance and had multiplicative effects with embB mutations on MICs. Ethambutol resistance is acquired through the acquisition of mutations that interact in complex ways to produce a range of MICs, from those falling below breakpoint values to ones representing high-level resistance. [ABSTRACT FROM AUTHOR]

We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis by the previously uncharacterized mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser55 site in Pks13 conferred TP resistance in M. tuberculosis. Overexpression of wild-type Pks13 resulted in TP resistance, and overexpression of the Pks13F79S mutant conferred high resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type M. tuberculosis, but it did so to a much lesser extent in TP-resistant M. tuberculosis. TP treatment was bactericidal and equivalent to treatment with the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment resulted in sterilizing activity. Computational docking identified a possible TP-binding groove within the Pks13 acyl carrier protein domain. This study confirms that M. tuberculosis Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway. [ABSTRACT FROM AUTHOR]

Little is known about the physiology of latent Mycobacterium tuberculosis infection. We studied the mutational rates of 24 index tuberculosis (TB) cases and their latently infected household contacts who developed active TB up to 5.25 years later, as an indication of bacterial physiological state and possible generation times during latent TB infection in humans. Here we report that the rate of new mutations in the M. tuberculosis genome decline dramatically after two years of latent infection (two-sided p < 0.001, assuming an 18 h generation time equal to log phase M. tuberculosis, with latency period modeled as a continuous variable). Alternatively, assuming a fixed mutation rate, the generation time increases over the latency duration. Mutations indicative of oxidative stress do not increase with increasing latency duration suggesting a lack of host or bacterial derived mutational stress. These results suggest that M. tuberculosis enters a quiescent state during latency, decreasing the risk for mutational drug resistance and increasing generation time, but potentially increasing bacterial tolerance to drugs that target actively growing bacteria. Here, Colangeli et al. use a human household contact cohort to measure the rate of mutation and evolution of Mycobacterium tuberculosis strains and find that long term latency is characterized by reduced mutation rates compared to latency in the first year, coinciding with clinical risk of developing active TB. [ABSTRACT FROM AUTHOR]

Isoniazid is one of the most effective antituberculosis drugs, yet its precise mechanism of action is still controversial. Using specialized linkage transduction, a single point mutation allele (S94A) within the putative target gene inhA was transferred in Mycobacterium tuberculosis. The inhA(S94A) allele was sufficient to confer clinically relevant levels of resistance to isoniazid killing and inhibition of mycolic acid biosynthesis. This resistance correlated with the decreased binding of the INH-NAD inhibitor to InhA, as shown by enzymatic and X-ray crystallographic analyses, and establishes InhA as the primary target of isoniazid action in M. tuberculosis. [ABSTRACT FROM AUTHOR]

Drug-resistant tuberculosis poses a persistent public health threat. The ReSeqTB platform is a collaborative, curated knowledgebase, designed to standardize and aggregate global Mycobacterium tuberculosis complex (MTBC) variant data from whole genome sequencing (WGS) with phenotypic drug susceptibility testing (DST) and clinical data. We developed a unified analysis variant pipeline (UVP) (https://github.com/CPTR-ReSeqTB/UVP) to identify variants and assign lineage from MTBC sequence data. Stringent thresholds and quality control measures were incorporated in this open source tool. The pipeline was validated using a well-characterized dataset of 90 diverse MTBC isolates with conventional DST and DNA Sanger sequencing data. The UVP exhibited 98.9% agreement with the variants identified using Sanger sequencing and was 100% concordant with conventional methods of assigning lineage. We analyzed 4636 publicly available MTBC isolates in the ReSeqTB platform representing all seven major MTBC lineages. The variants detected have an above 94% accuracy of predicting drug based on the accompanying DST results in the platform. The aggregation of variants over time in the platform will establish confidence-graded mutations statistically associated with phenotypic drug resistance. These tools serve as critical reference standards for future molecular diagnostic assay developers, researchers, public health agencies and clinicians working towards the control of drug-resistant tuberculosis. [ABSTRACT FROM AUTHOR]