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1.
Emerg Microbes Infect ; 9(1): 1342-1353, 2020 Dec.
Article in English | MEDLINE | ID: mdl-32538300

ABSTRACT

The Beijing genotype comprises a highly disseminated strain type that is frequently associated with multidrug resistant (MDR) tuberculosis (TB) and increased transmissibility but, countries such as Portugal and Guinea-Bissau fall outside the regions phylogeographically associated with this specific genotype. Nevertheless, recent data shows that this genotype might be gradually emerging in these two countries as an underlying cause of primary MDR-TB. Here, we describe the emergence of Mycobacterium tuberculosis Beijing strains associated with MDR-TB in Portugal and Guinea-Bissau demonstrating the presence of the well described superclusters 100-32 and 94-32 in Portugal and Guinea-Bissau, respectively. Genome-wide analysis and comparison with a global genomic dataset of M. tuberculosis Beijing strains, revealed the presence of two genomic clusters encompassing isolates from Portugal and Guinea-Bissau, GC1 (n = 121) and GC2 (n = 39), both of which bore SNP signatures compatible with the 100-32/B0/W148 and 94-32/Central Asia Outbreak clades, respectively. Moreover, GC2 encompasses a cross-border cluster between Portugal, Guinea-Bissau and Brazil thus supporting migration-associated introduction of MDR-TB and subsequent clonal expansion at the community-level. The comparison with global Beijing datasets demonstrates the global reach of the disease and its complex dissemination across multiple countries while in parallel there are clear microevolutionary trajectories towards extensively drug resistant TB.


Subject(s)
DNA, Bacterial/genetics , Drug Resistance, Multiple, Bacterial , Mycobacterium tuberculosis/classification , Tuberculosis, Multidrug-Resistant/microbiology , Whole Genome Sequencing/methods , Beijing , Brazil , Guinea-Bissau , High-Throughput Nucleotide Sequencing , Humans , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Phylogeny , Phylogeography , Portugal
2.
Sci Rep ; 10(1): 2600, 2020 02 13.
Article in English | MEDLINE | ID: mdl-32054988

ABSTRACT

Portugal is a low incidence country for tuberculosis (TB) disease. Now figuring among TB low incidence countries, it has since the 1990s reported multidrug resistant and extensively drug resistant (XDR) TB cases, driven predominantly by two strain-types: Lisboa3 and Q1. This study describes the largest characterization of the evolutionary trajectory of M/XDR-TB strains in Portugal, spanning a time-period of two decades. By combining whole-genome sequencing and phenotypic susceptibility data for 207 isolates, we report the geospatial patterns of drug resistant TB, particularly the dispersion of Lisboa3 and Q1 clades, which underly 64.2% and 94.0% of all MDR-TB and XDR-TB isolates, respectively. Genomic-based similarity and a phylogenetic analysis revealed multiple clusters (n = 16) reflecting ongoing and uncontrolled recent transmission of M/XDR-TB, predominantly associated with the Lisboa3 and Q1 clades. These clades are now thought to be evolving in a polycentric mode across multiple geographical districts. The inferred evolutionary history is compatible with MDR- and XDR-TB originating in Portugal in the 70's and 80's, respectively, but with subsequent multiple emergence events of MDR and XDR-TB particularly involving the Lisboa3 clade. A SNP barcode was defined for Lisboa3 and Q1 and comparison with a phylogeny of global strain-types (n = 28 385) revealed the presence of Lisboa3 and Q1 strains in Europe, South America and Africa. In summary, Portugal displays an unusual and unique epidemiological setting shaped by >40 years of uncontrolled circulation of two main phylogenetic clades, leading to a sympatric evolutionary trajectory towards XDR-TB with the potential for global reach.


Subject(s)
Extensively Drug-Resistant Tuberculosis/epidemiology , Extensively Drug-Resistant Tuberculosis/microbiology , Genome, Bacterial , Mycobacterium tuberculosis/genetics , Genetic Variation , Humans , Mycobacterium tuberculosis/isolation & purification , Phylogeny , Polymorphism, Single Nucleotide , Portugal/epidemiology , Whole Genome Sequencing
3.
BMC Genomics ; 20(1): 252, 2019 Mar 29.
Article in English | MEDLINE | ID: mdl-30922221

ABSTRACT

BACKGROUND: Continuing evolution of the Mycobacterium tuberculosis (Mtb) complex genomes associated with resistance to anti-tuberculosis drugs is threatening tuberculosis disease control efforts. Both multi- and extensively drug resistant Mtb (MDR and XDR, respectively) are increasing in prevalence, but the full set of Mtb genes involved are not known. There is a need for increased sensitivity of genome-wide approaches in order to elucidate the genetic basis of anti-microbial drug resistance and gain a more detailed understanding of Mtb genome evolution in a context of widespread antimicrobial therapy. Population structure within the Mtb complex, due to clonal expansion, lack of lateral gene transfer and low levels of recombination between lineages, may be reducing statistical power to detect drug resistance associated variants. RESULTS: To investigate the effect of lineage-specific effects on the identification of drug resistance associations, we applied genome-wide association study (GWAS) and convergence-based (PhyC) methods to multiple drug resistance phenotypes of a global dataset of Mtb lineages 2 and 4, using both lineage-wise and combined approaches. We identify both well-established drug resistance variants and novel associations; uniquely identifying associations for both lineage-specific and -combined GWAS analyses. We report 17 potential novel associations between antimicrobial resistance phenotypes and Mtb genomic variants. CONCLUSIONS: For GWAS, both lineage-specific and -combined analyses are useful, whereas PhyC may perform better in contexts of greater diversity. Unique associations with XDR in lineage-specific analyses provide evidence of diverging evolutionary trajectories between lineages 2 and 4 in response to antimicrobial drug therapy.


Subject(s)
Genome-Wide Association Study/methods , Mycobacterium tuberculosis/genetics , Polymorphism, Genetic , Tuberculosis, Multidrug-Resistant , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial , Evolution, Molecular , Gene Transfer, Horizontal , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Whole Genome Sequencing
5.
Nat Genet ; 50(2): 307-316, 2018 02.
Article in English | MEDLINE | ID: mdl-29358649

ABSTRACT

To characterize the genetic determinants of resistance to antituberculosis drugs, we performed a genome-wide association study (GWAS) of 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries. A GWAS approach within a mixed-regression framework was followed by a phylogenetics-based test for independent mutations. In addition to mutations in established and recently described resistance-associated genes, novel mutations were discovered for resistance to cycloserine, ethionamide and para-aminosalicylic acid. The capacity to detect mutations associated with resistance to ethionamide, pyrazinamide, capreomycin, cycloserine and para-aminosalicylic acid was enhanced by inclusion of insertions and deletions. Odds ratios for mutations within candidate genes were found to reflect levels of resistance. New epistatic relationships between candidate drug-resistance-associated genes were identified. Findings also suggest the involvement of efflux pumps (drrA and Rv2688c) in the emergence of resistance. This study will inform the design of new diagnostic tests and expedite the investigation of resistance and compensatory epistatic mechanisms.


Subject(s)
Drug Resistance, Multiple, Bacterial/genetics , Extensively Drug-Resistant Tuberculosis/microbiology , Genome, Bacterial , Mycobacterium tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/microbiology , Antitubercular Agents/therapeutic use , DNA, Bacterial/analysis , Extensively Drug-Resistant Tuberculosis/drug therapy , Genetic Variation , Genome-Wide Association Study , Geography , Humans , Microbial Sensitivity Tests , Mutation , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/isolation & purification , Phylogeny , Sequence Analysis, DNA , Tuberculosis, Multidrug-Resistant/drug therapy
6.
BMC Genomics ; 17: 151, 2016 Feb 29.
Article in English | MEDLINE | ID: mdl-26923687

ABSTRACT

BACKGROUND: Approximately 10% of the Mycobacterium tuberculosis genome is made up of two families of genes that are poorly characterized due to their high GC content and highly repetitive nature. The PE and PPE families are typified by their highly conserved N-terminal domains that incorporate proline-glutamate (PE) and proline-proline-glutamate (PPE) signature motifs. They are hypothesised to be important virulence factors involved with host-pathogen interactions, but their high genetic variability and complexity of analysis means they are typically disregarded in genome studies. RESULTS: To elucidate the structure of these genes, 518 genomes from a diverse international collection of clinical isolates were de novo assembled. A further 21 reference M. tuberculosis complex genomes and long read sequence data were used to validate the approach. SNP analysis revealed that variation in the majority of the 168 pe/ppe genes studied was consistent with lineage. Several recombination hotspots were identified, notably pe_pgrs3 and pe_pgrs17. Evidence of positive selection was revealed in 65 pe/ppe genes, including epitopes potentially binding to major histocompatibility complex molecules. CONCLUSIONS: This, the first comprehensive study of the pe and ppe genes, provides important insight into M. tuberculosis diversity and has significant implications for vaccine development.


Subject(s)
Genes, Bacterial , Multigene Family , Mycobacterium tuberculosis/genetics , Polymorphism, Single Nucleotide , Recombination, Genetic , DNA, Bacterial/genetics , Evolution, Molecular , Genome, Bacterial , Genomics/methods , Genotype , Mutation , Phylogeny , Selection, Genetic , Sequence Analysis, DNA
7.
Genome Med ; 7(1): 51, 2015.
Article in English | MEDLINE | ID: mdl-26019726

ABSTRACT

Mycobacterium tuberculosis drug resistance (DR) challenges effective tuberculosis disease control. Current molecular tests examine limited numbers of mutations, and although whole genome sequencing approaches could fully characterise DR, data complexity has restricted their clinical application. A library (1,325 mutations) predictive of DR for 15 anti-tuberculosis drugs was compiled and validated for 11 of them using genomic-phenotypic data from 792 strains. A rapid online 'TB-Profiler' tool was developed to report DR and strain-type profiles directly from raw sequences. Using our DR mutation library, in silico diagnostic accuracy was superior to some commercial diagnostics and alternative databases. The library will facilitate sequence-based drug-susceptibility testing.

8.
Infect Genet Evol ; 18: 238-46, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23648425

ABSTRACT

Human tuberculosis is an infectious disease caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). Although spoligotyping and MIRU-VNTR are standard methodologies in MTBC genetic epidemiology, recent studies suggest that Single Nucleotide Polymorphisms (SNP) are advantageous in phylogenetics and strain group/lineages identification. In this work we use a set of 79 SNPs to characterize 1987 MTBC isolates from Portugal and 141 from Northeast Brazil. All Brazilian samples were further characterized using spolygotyping. Phylogenetic analysis against a reference set revealed that about 95% of the isolates in both populations are singly attributed to bacterial lineage 4. Within this lineage, the most frequent strain groups in both Portugal and Brazil are LAM, followed by Haarlem and X. Contrary to these groups, strain group T showed a very different prevalence between Portugal (10%) and Brazil (1.5%). Spoligotype identification shows about 10% of mis-matches compared to the use of SNPs and a little more than 1% of strains unidentifiability. The mis-matches are observed in the most represented groups of our sample set (i.e., LAM and Haarlem) in almost the same proportion. Besides being more accurate in identifying strain groups/lineages, SNP-typing can also provide phylogenetic relationships between strain groups/lineages and, thus, indicate cases showing phylogenetic incongruence. Overall, the use of SNP-typing revealed striking similarities between MTBC populations from Portugal and Brazil.


Subject(s)
Mycobacterium tuberculosis/genetics , Tuberculosis/epidemiology , Tuberculosis/microbiology , Brazil/epidemiology , Cluster Analysis , DNA, Bacterial/genetics , DNA, Intergenic/genetics , Humans , Molecular Epidemiology , Molecular Typing , Mycobacterium tuberculosis/classification , Phylogeny , Polymorphism, Single Nucleotide , Portugal/epidemiology
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