TBPore Cluster: A Novel Phylogenetic Pipeline for Mycobacterium tuberculosis Transmission Studies Using Nanopore Data
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ABSTRACT:
ORGANISM(S): Mycobacterium tuberculosis complex
SUBMITTER:
PROVIDER: S-BSST1485 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Purpose: Tuberculosis is still a major threat to global health. New tools and strategies to produce disease-related proteins are quintessential for the development of novel vaccines and diagnostic markers. Experimental design: To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. The potential for the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays. Results: Twelve soluble proteins from Mtb, including one protein complex, were purified to homogeneity following recombinant expression in M. smegmatis. Protein purity was assessed both by size exclusion chromatography and mass spectrometry. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients. Conclusion and clinical relevance: The established workflow comprises a simple, cost-effective and scalable pipeline for production of soluble proteins from Mtb and can be used to prioritize immunogenic proteins suitable for use as diagnostic markers.
2018-06-27 | PXD004133 | Pride
Project description:Mycobacterium tuberculosis Beijing genotype originated in China and has undergone a dramatic population growth and global spread in the last century. Here, a collection of M. tuberculosis Beijing family isolates from different provinces across all China was genotyped by high-resolution (24-MIRU-VNTR) and low-resolution, high-rank (modern and ancient sublineages) markers. The molecular profiles and global and local phylogenies were compared to the strain phenotype and patient data. The phylogeographic patterns observed in the studied collection demonstrate that large-scale (but not middle/small-scale) distance remains one of the decisive factors of the genetic divergence of M. tuberculosis populations. Analysis of diversity and network topology of the local collections appears to corroborate a recent intriguing hypothesis about Beijing genotype originating in South China. Placing our results within the Eurasian context suggested that important Russian B0/W148 and Asian/Russian A0/94-32 epidemic clones of the Beijing genotype could trace their origins to the northeastern and northwestern regions of China, respectively. The higher clustering of the modern isolates in children and lack of increased MDR rate in any sublineage suggest that not association with drug resistance but other (e.g., speculatively, virulence-related) properties underlie an enhanced dissemination of the evolutionarily recent, modern sublineage of the Beijing genotype in China.
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Project description:Widespread and frequent resistance to the second-line tuberculosis (TB) medicine streptomycin, suggests ongoing transmission of low fitness cost streptomycin resistance mutations. To investigate this hypothesis, we studied a cohort of 681 individuals from a TB epidemic in Portugal. Whole-genome sequencing (WGS) analyses were combined with phenotypic growth studies in culture media and in mouse bone marrow derived macrophages. Streptomycin resistance was the most frequent resistance in the cohort accounting for 82.7% (n = 67) of the resistant Mycobacterium tuberculosis isolates. WGS of 149 clinical isolates identified 13 transmission clusters, including three clusters containing only streptomycin resistant isolates. The biggest cluster was formed by eight streptomycin resistant isolates with a maximum of five pairwise single nucleotide polymorphisms of difference. Interestingly, despite their genetic similarity, these isolates displayed different resistance levels to streptomycin, as measured both in culture media and in infected mouse bone marrow derived macrophages. The genetic bases underlying this phenotype are a combination of mutations in gid and other genes. This study suggests that specific streptomycin resistance mutations were transmitted in the cohort, with the resistant isolates evolving at the cluster level to allow low-to-high streptomycin resistance levels without a significative fitness cost. This is relevant not only to better understand transmission of streptomycin resistance in a clinical setting dominated by Lineage 4 M. tuberculosis infections, but mainly because it opens new prospects for the investigation of selection and spread of drug resistance in general.
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Project description:Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of the rate of change in the genome over relevant timescales is required.We attempted to estimate a molecular clock by sequencing 199 isolates from epidemiologically linked tuberculosis cases, collected in the Netherlands spanning almost 16 years.Multiple analyses support an average mutation rate of ~0.3 SNPs per genome per year. However, all analyses revealed a very high degree of variation around this mean, making the confirmation of links proposed by epidemiology, and inference of novel links, difficult. Despite this, in some cases, the phylogenetic context of other strains provided evidence supporting the confident exclusion of previously inferred epidemiological links.This in-depth analysis of the molecular clock revealed that it is slow and variable over short time scales, which limits its usefulness in transmission studies. However, the superior resolution of whole genome sequencing can provide the phylogenetic context to allow the confident exclusion of possible transmission events previously inferred via traditional DNA fingerprinting techniques and epidemiological cluster investigation. Despite the slow generation of variation even at the whole genome level we conclude that the investigation of tuberculosis transmission will benefit greatly from routine whole genome sequencing.
| S-EPMC3599118 | biostudies-literature
Project description:Genotyping tools help identify the complexity in Mycobacterium tuberculosis transmission clusters. We carried out a thorough analysis of the epidemiological and bacteriological complexity of a cluster in Almería, Spain. The cluster, initially associated with Moroccan migrants and with no secondary cases identified in 4 years, then reappeared in Spanish-born individuals. In one case, two Mycobacterium tuberculosis clonal variants were identified. We reanalyzed the cluster, supported by the characterization of multiple cultured isolates and respiratory specimens, whole-genome sequencing, and epidemiological case interviews. Our findings showed that the cluster, which was initially thought to have restarted activity with just a single case harboring a small degree of within-host diversity, was in fact currently growing due to coincidental reactivation of past exposures, with clonal diversity transmitted throughout the cluster. In one case, within-host diversity was amplified, probably due to prolonged diagnostic delay. IMPORTANCE The precise study of the dynamics of tuberculosis transmission in socio-epidemiologically complex scenarios may require more thorough analysis than the standard molecular epidemiology strategies. Our study illustrates the epidemiological and bacteriological complexity present in a transmission cluster in a challenging epidemiological setting with a high proportion of migrant cases. The combination of whole-genome sequencing, refined and refocused epidemiological interviews, and in-depth analysis of the bacterial composition of sputa and cultured isolates was crucial in order to correctly reinterpret the true nature of this cluster. Our global approach allowed us to reinterpret correctly the unnoticed epidemiological and bacteriological complexity involved in the Mycobacterium tuberculosis transmission event under study, which had been overlooked by the usual molecular epidemiology approaches.
| S-EPMC8768656 | biostudies-literature
Project description:Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S](2+) cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S](+) state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5'-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5'-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.
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Project description:In the case of tuberculosis (TB), the capabilities of epidemic models to produce quantitatively robust forecasts are limited by multiple hindrances. Among these, understanding the complex relationship between disease epidemiology and populations' age structure has been highlighted as one of the most relevant. TB dynamics depends on age in multiple ways, some of which are traditionally simplified in the literature. That is the case of the heterogeneities in contact intensity among different age strata that are common to all airborne diseases, but still typically neglected in the TB case. Furthermore, while demographic structures of many countries are rapidly aging, demographic dynamics are pervasively ignored when modeling TB spreading. In this work, we present a TB transmission model that incorporates country-specific demographic prospects and empirical contact data around a data-driven description of TB dynamics. Using our model, we find that the inclusion of demographic dynamics is followed by an increase in the burden levels predicted for the next decades in the areas of the world that are most hit by the disease today. Similarly, we show that considering realistic patterns of contacts among individuals in different age strata reshapes the transmission patterns reproduced by the models, a result with potential implications for the design of age-focused epidemiological interventions.
| S-EPMC5889657 | biostudies-literature
Project description:Metagenomic sequencing combined with Oxford Nanopore Technology has the potential to become a point-of-care test for infectious disease in public health and clinical settings, providing rapid diagnosis of infection, guiding individual patient management and treatment strategies, and informing infection prevention and control practices. However, publicly available, streamlined, and reproducible pipelines for analyzing Nanopore metagenomic sequencing data are still lacking. Here we introduce NanoSPC, a scalable, portable and cloud compatible pipeline for analyzing Nanopore sequencing data. NanoSPC can identify potentially pathogenic viruses and bacteria simultaneously to provide comprehensive characterization of individual samples. The pipeline can also detect single nucleotide variants and assemble high quality complete consensus genome sequences, permitting high-resolution inference of transmission. We implement NanoSPC using Nextflow manager within Docker images to allow reproducibility and portability of the analysis. Moreover, we deploy NanoSPC to our scalable pathogen pipeline platform, enabling elastic computing for high throughput Nanopore data on HPC cluster as well as multiple cloud platforms, such as Google Cloud, Amazon Elastic Computing Cloud, Microsoft Azure and OpenStack. Users could either access our web interface (https://nanospc.mmmoxford.uk) to run cloud-based analysis, monitor process, and visualize results, as well as download Docker images and run command line to analyse data locally.
| S-EPMC7319573 | biostudies-literature
Project description:BackgroundAlthough Ethiopia ranks seventh among the world's 22 high-burden tuberculosis (TB) countries, little is known about strain diversity and transmission. In this study, we present the first in-depth analysis of the population structure and transmission dynamics of Mycobacterium tuberculosis strains from Northwest Ethiopia.MethodsIn the present study, 244 M. tuberculosis isolates where analysed by mycobacterial interspersed repetitive unit - variable number tandem repeat 24-loci typing and spoligotyping methods to determine phylogenetic lineages and perform cluster analysis. Clusters of strains with identical genotyping patterns were considered as an indicator for the recent transmission.ResultsOf 244 isolates, 59.0% were classified into nine previously described lineages: Dehli/CAS (38.9%), Haarlem (8.6%), Ural (3.3%), LAM (3.3%), TUR (2.0%), X-type (1.2%), S-type (0.8%), Beijing (0.4%) and Uganda II (0.4%). Interestingly, 31.6% of the strains were grouped into four new lineages and were named as Ethiopia_3 (13.1%), Ethiopia_1 (7.8%), Ethiopia_H37Rv like (7.0%) and Ethiopia_2 (3.7%) lineages. The remaining 9.4% of the isolates could not be assigned to the known or new lineages. Overall, 45.1% of the isolates were grouped in clusters, indicating a high rate of recent transmission.ConclusionsThis study confirms a highly diverse M. tuberculosis population structure, the presence of new phylogenetic lineages and a predominance of the Dehli/CAS lineage in Northwest Ethiopia. The high rate of recent transmission indicates defects of the TB control program in Northwest Ethiopia. This emphasizes the importance of strengthening laboratory diagnosis of TB, intensified case finding and treatment of TB patients to interrupt the chain of transmission.
| S-EPMC3605317 | biostudies-literature
Project description:Tuberculosis is still a major threat to global health. New tools and strategies to produce disease-related proteins are quintessential for the development of novel vaccines and diagnostic markers.To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. The potential for the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays.Twelve soluble proteins from Mtb, including one protein complex, were purified to near-homogeneity following recombinant expression in M. smegmatis. Protein purity was assessed both by size exclusion chromatography and MS. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients.The established workflow comprises a simple, cost-effective, and scalable pipeline for production of soluble proteins from Mtb and can be used to prioritize immunogenic proteins suitable for use as diagnostic markers.
| S-EPMC5095800 | biostudies-literature