Dataset Information

Spoligotyping and whole-genome sequencing analysis of lineage 1 strains of Mycobacterium tuberculosis in Da Nang, Vietnam.

ABSTRACT: Spacer oligonucleotide typing (spoligotyping), a widely used, classical genotyping method for Mycobacterium tuberculosis complex (MTBC), is a PCR-based dot-blot hybridization technique to detect the genetic diversity of the direct repeat (DR) region. Of the seven major MTBC lineages in the world, lineage 1 (Indo-Oceanic) mostly corresponds to the East African-Indian (EAI) spoligotype family in East Africa and Southeast Asia.We investigated the genomic features of Vietnamese lineage 1 strains, comparing spoligotype patterns using whole-genome sequencing (WGS) data.M. tuberculosis strains isolated in Da Nang, Vietnam were subjected to conventional spoligotyping, followed by WGS analysis using a high-throughput sequencer. Vietnamese lineage 1 strains were further analyzed with other lineage 1 strains obtained from a public database.Indicating a major spoligotype in Da Nang, 86 (46.2%) of the 186 isolates belonged to the EAI family or lineage 1. Although typical EAI4-VNM strains are characterized by the deletion of spacers 26 and 27, 65 (75.6%) showed ambiguous signals on spacer 26. De novo assembly of the entire DR region and in silico spoligotyping analysis suggested the absence of spacer 26, and direct sequencing revealed that the 17th spacer sequence not used for conventional typing, was cross-hybridized to the spacer 26 probe. Vietnamese EAI4-VNM, other EAI-like strains, and those showing a non-EAI pattern lacking many spacers formed a monophyletic group separate from other EAI families in the world.Information about the alignment of spacers in the entire DR region obtained from WGS data provides a clue for the determination of experimentally ambiguous spoligo patterns. WGS data also helped to analyze the hidden relationships between apparently distinct spoligo patterns.

SUBMITTER: Hijikata M

PROVIDER: S-EPMC5648229 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Spoligotyping and whole-genome sequencing analysis of lineage 1 strains of Mycobacterium tuberculosis in Da Nang, Vietnam.

Hijikata Minako M Keicho Naoto N Duc Le Van LV Maeda Shinji S Hang Nguyen Thi Le NTL Matsushita Ikumi I Kato Seiya S

PloS one 20171019 10

<h4>Background</h4>Spacer oligonucleotide typing (spoligotyping), a widely used, classical genotyping method for Mycobacterium tuberculosis complex (MTBC), is a PCR-based dot-blot hybridization technique to detect the genetic diversity of the direct repeat (DR) region. Of the seven major MTBC lineages in the world, lineage 1 (Indo-Oceanic) mostly corresponds to the East African-Indian (EAI) spoligotype family in East Africa and Southeast Asia.<h4>Objectives</h4>We investigated the genomic featur ...[more]

PMID: 29049400

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Project description:BackgroundTuberculosis (TB) is a serious problem in China. While there have been some studies on the nationwide genotyping of Mycobacterium tuberculosis (M. tuberculosis), there has been little detailed research in Beijing, the capital of China, which has a huge population. Here, M. tuberculosis clinical strains collected in Beijing during 2009 were genotyped by classical methods.Methodology/principal findingsOur aim was to analyze the genetic diversity of M. tuberculosis strains within the Beijing metropolitan area. We characterized these strains using two standard methods, spoligotyping (n?=?1585) and variable number of tandem repeat (VNTR) typing (n?=?1053). We found that the most prominent genotype was Beijing family genotype. Other genotypes included the MANU, T and H families etc. Spoligotyping resulted in 137 type patterns, included 101 unclustered strains and 1484 strains clustered into 36 clusters. In VNTR typing analysis, we selected 12-locus (QUB-11b, MIRU10, Mtub21, MIRU 23, MIRU39, MIRU16, MIRU40, MIRU31, Mtub24, Mtub04, MIRU20, and QUB-4156c) and named it 12-locus (BJ) VNTR. VNTR resulted in 869 type patterns, included 796 unclustered strains and 257 strains clustered into 73 clusters. It has almost equal discriminatory power to the 24-locus VNTR.Conclusions/significanceOur study provides a detailed characterization of the genotypic diversity of M. tuberculosis in Beijing. Combining spoligotyping and VNTR typing to study the genotyping of M. tuberculosis gave superior results than when these techniques were used separately. Our results indicated that Beijing family strains were still the most prevalent M. tuberculosis in Beijing. Moreover, VNTR typing analyzing of M. tuberculosis strains in Beijing was successfully accomplished using 12-locus (BJ) VNTR. This method used for strains genotyping from the Beijing metropolitan area was comparable. This study will not only provide TB researchers with valuable information for related studies, but also provides guidance for the prevention and control of TB in Beijing.

Project description:Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains represent a major threat for tuberculosis (TB) control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR) variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS) to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A) and nine (Patient B) polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and molecular drug resistance tests. Furthermore, high resolution WGS analysis is necessary to accurately detect exogenous re-infection as classical genotyping lacks discriminatory power in high incidence settings.

Dataset Information

Spoligotyping and whole-genome sequencing analysis of lineage 1 strains of Mycobacterium tuberculosis in Da Nang, Vietnam.

Publications

Spoligotyping and whole-genome sequencing analysis of lineage 1 strains of Mycobacterium tuberculosis in Da Nang, Vietnam.

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