Whole Genome Sequencing Of Mycobacterium Tuberculosis Research Articles

PCR Development for Analysis of Some Type II Toxin-Antitoxin Systems, relJK, mazEF3, and vapBC3 Genes, in Mycobacterium tuberculosis and Mycobacterium bovis.

Toxin-Antitoxin (TA) systems are some small genetic modules in bacteria that play significant roles in resistance and tolerance development to antibiotics. Whole genome sequencing (WGS) is an effective method to analyze TA systems in pathogenic Mycobacteria. However, this study aimed to use a simple and inexpensive PCR-Sequencing approach to investigate the type II TA system. Using data from the WGS of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv and Mycobacterium bovis (M. bovis) strain BCG, primers specific to the relJK, mazEF3, and vapBC3 gene families were designed by Primer3 software. Following that, a total of 90 isolates were examined using the newly developed PCR assay, consisting of 64M. tuberculosis and 26M. bovis isolates, encompassing both 45 rifampin-sensitive and 45 rifampin-resistant strains. Finally, 28 isolates (including 14 rifampin-resistant isolates) were sent for sequencing, and their sequences were aligned and compared to the mentioned reference sequences. The amplicons size of mazEF3, relJK, and vapBC3 genes were 825, 875, and 934 bp, respectively. Furthermore, all tested isolates showed the specific amplicons for these TA families. To evaluate the specificity of the primers, PCR was performed on S. aureus and E.coli isolates. None of the examined samples had the desired amplicons. Therefore, the primers had acceptable specificity. The results indicated that the developed PCR-Sequencing approach can be used to effectively investigate certain types of TA systems. Considering high costs of WGS and difficulty in interpreting its results, such a simple and inexpensive method is beneficial in the evaluation of TA systems in Mycobacteria.

Impact of Whole-Genome Sequencing of Mycobacterium tuberculosis on Treatment Outcomes for MDR-TB/XDR-TB: A Systematic Review.

Five electronic databases were searched for studies published in English from the year 2000 onward. Two reviewers independently conducted the article screening, relevant data extraction, and quality assessment. The data of the included studies were synthesized with a narrative method and are presented in a tabular format. The database search identified 949 published articles and 8 studies were included. An unfavorable treatment outcome was reported for 26.6% (488/1834) of TB cases, which ranged from 9.7 to 51.3%. Death was reported in 10.5% (194/1834) of total cases. High-level fluoroquinolone resistance (due to gyrA 94AAC and 94GGC mutations) was correlated as the cause of unfavorable treatment outcomes and reported in three studies. Other drug resistance mutations, like kanamycin high-level resistance mutations (rrs 1401G), rpoB Ile491Phe, and ethA mutations, conferring prothionamide resistance were also reported. The secondary findings from this systematic review involved laboratory aspects of WGS, including correlations with phenotypic DST, cost, and turnaround time, or the impact of WGS results on public health actions, such as determining transmission events within outbreaks. WGS has a significant capacity to provide accurate and comprehensive drug resistance data for MDR/XDR-TB, which can inform personalized drug therapy to optimize treatment outcomes.

Implementation of national whole-genome sequencing of Mycobacterium tuberculosis, National Public Health Laboratory, Singapore, 2019-2022.

The National Tuberculosis Programme (NTBP) monitors the occurrence and spread of tuberculosis (TB) and multidrug-resistant TB (MDR-TB) in Singapore. Since 2020, whole-genome sequencing (WGS) of Mycobacterium tuberculosis isolates has been performed at the National Public Health Laboratory (NPHL) for genomic surveillance, replacing spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeats analysis (MIRU-VNTR). Four thousand three hundred and seven samples were sequenced from 2014 to January 2023, initially as research projects and later developed into a comprehensive public health surveillance programme. Currently, all newly diagnosed culture-positive cases of TB in Singapore are prospectively sent for WGS, which is used to perform lineage classification, predict drug resistance profiles and infer genetic relationships between TB isolates. This paper describes NPHL's operational and technical experiences with implementing the WGS service in an urban TB-endemic setting, focusing on cluster detection and genomic drug susceptibility testing (DST). Cluster detection: WGS has been used to guide contact tracing by detecting clusters and discovering unknown transmission networks. Examples have been clusters in a daycare centre, housing apartment blocks and a horse-racing betting centre. Genomic DST: genomic DST prediction (gDST) identifies mutations in core genes known to be associated with TB drug resistance catalogued in the TBProfiler drug resistance mutation database. Mutations are reported with confidence scores according to a standardized approach referencing NPHL's internal gDST confidence database, which is adapted from the World Health Organization (WHO) TB drug mutation catalogue. Phenotypic-genomic concordance was observed for the first-line drugs ranging from 2959/2998 (98.7 %) (ethambutol) to 2983/2996 (99.6 %) (rifampicin). Aspects of internal database management, reporting standards and caveats in results interpretation are discussed.

Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates from Diabetic and Non-Diabetic Patients with Pulmonary Tuberculosis.

The association of tuberculosis and type 2 diabetes mellitus has been a recognized re-emerging challenge in management of the convergence of the two epidemics. Though much of the literature has studied this association, there is less knowledge in the field of genetic diversities that might occur in strains infecting tuberculosis patients with and without diabetes. Our study focused on determining the extent of diversity of genotypes of Mycobacterium tuberculosis in both these categories of patients. We subjected 55 M. tuberculosis isolates from patients diagnosed with pulmonary TB with and without type 2 diabetes mellitus to whole-genome sequencing on Illumina Hi Seq platform. The most common lineage identified was lineage 1, the Indo-Oceanic lineage (n = 22%), followed by lineage 4, the Euro-American lineage (n = 18, 33%); lineage 3, the East-African Indian lineage (n = 13, 24%); and lineage 2, the East-Asian lineage (n = 1, 2%). There were no significant differences in the distribution of lineages in both diabetics and non-diabetics in the South Indian population, and further studies involving computational analysis and comparative transcriptomics are needed to provide deeper insights.

An overview of tuberculosis outbreaks reported in the years 2011–2020

BackgroundIn many countries tuberculosis (TB) remains a highly prevalent disease and a major contributor to infectious disease mortality. The fight against TB requires surveillance of the population of strains circulating worldwide and the analysis of the prevalence of certain strains in populations. Nowadays, whole genome sequencing (WGS) allows for accurate tracking of TB transmission. Currently, there is a lack of a comprehensive summary of the characteristics of TB outbreaks.MethodsWe systematically analyzed studies reporting TB outbreaks worldwide, monitored through WGS of Mycobacterium tuberculosis. We 1) mapped the reported outbreaks from 2011- 2020, 2) estimated the average size of the outbreaks, 3) indicated genetic lineages causing the outbreaks, and 4) determined drug-resistance patterns of M. tuberculosis strains involved in the outbreaks.ResultsMost data originated from Europe, Asia, and North America. We found that TB outbreaks were reported throughout the globe, on all continents, and in countries with both high and low incidences. The detected outbreaks contained a median of five M. tuberculosis isolates. Most strains causing the outbreaks belonged to lineage four, more rarely to lineage two. Reported outbreak isolates were often drug resistant.ConclusionsWe conclude that more WGS surveillance of M. tuberculosis outbreaks is needed. Globally standardized procedures might improve the control of M. tuberculosis infections.

Whole-Genome Sequencing of Mycobacterium tuberculosis Isolated from Three Hospitals in South Korea.

Mycobacterium tuberculosis clinical isolates CN118, CN272, CB131, IP004, and IP016 were isolated from three hospitals in South Korea. Here, we report the whole-genome sequences of the five M. tuberculosis clinical isolates to aid the understanding of their genetic diversity.

Whole-genome sequencing of Mycobacterium tuberculosis from Cambodia

Cambodia has one of the highest tuberculosis (TB) incidence rates in the WHO Western Pacific region. Remarkably though, the prevalence of multidrug-resistant TB (MDR-TB) remains low. We explored the genetic diversity of Mycobacterium tuberculosis (MTB) circulating in this unique setting using whole-genome sequencing (WGS). From October 2017 until January 2018, we collected one hundred sputum specimens from consenting adults older than 21 years of age, newly diagnosed with bacteriologically confirmed TB in 3 districts of Phnom Penh and Takeo provinces of Cambodia before they commence on their TB treatment, where eighty MTB isolates were successfully cultured and sequenced. Majority of the isolates belonged to Lineage 1 (Indo-Oceanic) (69/80, 86.25%), followed by Lineage 2 (East Asian) (10/80, 12.5%) and Lineage 4 (Euro-American) (1/80, 1.25%). Phenotypic resistance to both streptomycin and isoniazid was found in 3 isolates (3/80, 3.75%), while mono-resistance to streptomycin and isoniazid was identical at 2.5% (N = 2 each). None of the isolates tested was resistant to either rifampicin or ethambutol. The specificities of genotypic prediction for resistance to all drugs tested were 100%, while the sensitivities of genotypic resistance predictions to isoniazid and streptomycin were lower at 40% (2/5) and 80% (4/5) respectively. We identified 8 clusters each comprising of two to five individuals all residing in the Takeo province, making up half (28/56, 50%) of all individuals sampled in the province, indicating the presence of multiple ongoing transmission events. All clustered isolates were of Lineage 1 and none are resistant to any of the drugs tested. This study while demonstrating the relevance and utility of WGS in predicting drug resistance and inference of disease transmission, highlights the need to increase the representation of genotype–phenotype TB data from low and middle income countries in Asia and Africa to improve the accuracies for prediction of drug resistance.

Whole-Genome Sequencing of Mycobacterium tuberculosis Directly from Sputum Samples.

Whole-genome sequencing is a powerful, high-resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography, and mutations associated with antimicrobial resistance. The ability to sequence pathogen genomes directly from clinical specimens, without the requirement for in vitro culturing, is attractive in terms of time- and labor-saving, especially in the case of slow growing pathogens, such as Mycobacterium tuberculosis. However, clinical samples typically contain toolow levels of pathogen nucleic acid, plus relatively high levels of human and natural microbiota DNA/RNA, to make this a viable option. Using a combination of whole-genome enrichment and deep sequencing, which has been proven to be a nonmutagenic approach, we can capture all known variations found within M. tuberculosis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of M. tuberculosis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.

Whole-genome sequencing of Mycobacterium tuberculosis directly from clinical samples for high-resolution genomic epidemiology and drug resistance surveillance: an observational study

Direct whole-genome sequencing of Mycobacterium tuberculosis from clinical specimens will be a major breakthrough in tuberculosis diagnosis and control. To date, direct whole-genome sequencing has never been used in genomic epidemiology, and its accuracy in transmission inference remains unknown. We investigated the technical challenges imposed by direct whole-genome sequencing, and used it to infer transmission clusters and predict drug resistance. Using an optimised workflow, we did direct whole-genome sequencing for 37 clinical specimens from 23 tuberculosis patients. Nine sputum samples from nine patients who were infected with different non-tuberculous mycobacteria and culture-negative for tuberculosis were used as controls in the qPCR assays and pre-sequencing runs. Additionally, 780 clinical isolates in the region of Comunidad Valenciana (Spain) were whole-genome sequenced between Jan 1, 2014, and Dec 31, 2016. We analysed the genomic variants to build a tuberculosis transmission network for the region, including the clinical specimens, and to predict drug susceptibility profiles. After sequencing 37 clinical specimens, 28 specimens (22 [85%] of 26 smear-positive and six [55%] of 11 smear-negative) met the quality criteria for downstream analysis. All 28 clinical specimens clustered with their matching culture isolates, with a median distance of 0 single nucleotide polymorphisms. Of the 28 clinical specimens, 16 (57%) were accurately assigned to ten transmission clusters in the region, and 12 (43%) were unique cases. Transmission inferences and drug-susceptibility predictions from direct whole-genome sequencing data were concordant with sequences from corresponding cultures and phenotypic drug-susceptibility testing. Complete genomic analysis, within a week of specimen receipt, cost €217 per sample (excluding personnel costs). Direct whole-genome sequencing could be used to accurately delineate transmission clusters of tuberculosis and conduct culture-independent surveillance. Compared with conventional approaches, direct whole-genome sequencing allows researchers to do real-time genomic epidemiology and drug resistance surveillance in settings where culture and drug susceptibility testing are not available. European Research Council; Ministerio de Ciencia, Innovación y Universidades (Spanish Government).

Elucidation of drug resistance mutations in Mycobacterium tuberculosis isolates from North India by whole-genome sequencing.

Rapid diagnosis of drug-resistant tuberculosis (TB) is required for better patient management and treatment outcomes. Whole-genome sequencing (WGS) can be used to detect single nucleotide polymorphisms (SNPs) and deletions/insertions that are responsible for mostMycobacterium tuberculosis drug resistance. WGS is being performed at scale in high-income countries, but there are limited reports of its use in India. In this study, 33 clinicalM. tuberculosis isolates from the Mycobacterial Repository in Chandigarh underwent WGS. Phenotypic drug susceptibility testing was performed according to World Health Organization (WHO) recommendations. Four isolates were excluded from the analysis due to culture contamination or mislabelling during the study. Among the remaining 29 isolates, 21 (72.4%) were multidrug-resistant TB (MDR-TB) and 1 (3.4%) was extensively-drug resistant TB (XDR-TB). The most common mutations observed for isoniazid, rifampicin, ofloxacin and kanamycin resistance werekatG(S315T), rpoB(S450L), gyrA(A90V) and rrs(A1401G), respectively. The isolates mainly belonged to lineages 2 and 3, with most MDR-TB among lineage 2 isolates. WGS ofM. tuberculosis isolates allows the detection of drug resistance to all drugs in a single test and also provides insight into the evolution and drug-resistant TB.

Whole-Genome Sequencing of Mycobacterium tuberculosis Provides Insight into the Evolution and Genetic Composition of Drug-Resistant Tuberculosis in Belarus.

ABSTRACTThe emergence and spread of drug-resistant Mycobacterium tuberculosis (DR-TB) are critical global health issues. Eastern Europe has some of the highest incidences of DR-TB, particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. To better understand the genetic composition and evolution of MDR- and XDR-TB in the region, we sequenced and analyzed the genomes of 138 M. tuberculosis isolates from 97 patients sampled between 2010 and 2013 in Minsk, Belarus. MDR and XDR-TB isolates were significantly more likely to belong to the Beijing lineage than to the Euro-American lineage, and known resistance-conferring loci accounted for the majority of phenotypic resistance to first- and second-line drugs in MDR and XDR-TB. Using a phylogenomic approach, we estimated that the majority of MDR-TB was due to the recent transmission of already-resistant M. tuberculosis strains rather than repeated de novo evolution of resistance within patients, while XDR-TB was acquired through both routes. Longitudinal sampling of M. tuberculosis from 34 patients with treatment failure showed that most strains persisted genetically unchanged during treatment or acquired resistance to fluoroquinolones. HIV+ patients were significantly more likely to have multiple infections over time than HIV− patients, highlighting a specific need for careful infection control in these patients. These data provide a better understanding of the genomic composition, transmission, and evolution of MDR- and XDR-TB in Belarus and will enable improved diagnostics, treatment protocols, and prognostic decision-making.

Whole-genome sequencing of Mycobacterium tuberculosis for rapid diagnostics and beyond

Whole-genome sequencing of Mycobacterium tuberculosis for rapid diagnostics and beyond

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction.

The annotated whole-genome sequence of Mycobacterium tuberculosis indicated that Rv1388 (Mtihf) likely encodes a putative 20 kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or organization of mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF-duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg170, Arg171, and Arg173, which might be involved in DNA binding, and a conserved proline (P150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methylmethanesulfonate could be complemented with the wild-type Mtihf, but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, bind with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes compaction of DNA into nucleoid-like or higher-order filamentous structures. We hence propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.

Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples.

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5' overhangs relative to the 3' overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having ≥18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3' overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5' overhangs, it could also catalyze significant unwinding of substrates containing 3' overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5' → 3' and weak 3' → 5' unwinding activities. The extent of unwinding of Y-shaped DNA structures was ∼3-fold lower compared with duplexes with 5' overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.

Whole-genome sequencing of Mycobacterium tuberculosis as an epidemiological marker

Whole-genome sequencing of Mycobacterium tuberculosis as an epidemiological marker