Molecular Mycobacteriology

Borstel-Hohenraden, Germany

Molecular Mycobacteriology

Borstel-Hohenraden, Germany
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Niemann S.,Molecular Mycobacteriology | Niemann S.,German Center for Infection Research | Merker M.,Molecular Mycobacteriology | Kohl T.,Molecular Mycobacteriology | Supply P.,Lille University Hospital Center
Microbiology Spectrum | Year: 2016

Tuberculosis (TB) remains the most deadly bacterial infectious disease worldwide. Its treatment and control are threatened by increasing numbers of multidrug-resistant (MDR) or nearly untreatable extensively drug-resistant (XDR) strains. New concepts are therefore urgently needed to understand the factors driving the TB epidemics and the spread of different strain populations, especially in association with drug resistance. Classical genotyping and, more recently, whole-genome sequencing (WGS) revealed that the world population of tubercle bacilli is more diverse than previously thought. Several major phylogenetic lineages can be distinguished, which are associated with their sympatric host population. Distinct clonal (sub)populations can even coexist within infected patients. WGS is now used as the ultimate approach for differentiating clinical isolates and for linking phenotypic to genomic variation from lineage to strain levels. Multiple lines of evidence indicate that the genetic diversity of TB strains translates into pathobiological consequences, and key molecular mechanisms probably involved in differential pathoadaptation of some main lineages have recently been identified. Evidence also accumulates on molecular mechanisms putatively fostering the emergence and rapid expansion of particular MDR and XDR strain groups in some world regions. However, further integrative studies will be needed for complete elucidation of the mechanisms that allow the pathogen to infect its host, acquire multidrug resistance, and transmit so efficiently. Such knowledge will be key for the development of the most effective new diagnostics, drugs, and vaccination strategies. © 2016 American Society for Microbiology. All rights reserved.


Kohl T.A.,Molecular Mycobacteriology | Diel R.,University of Kiel | Harmsen D.,University of Munster | Rothganger J.,Ridom GmbH | And 4 more authors.
Journal of Clinical Microbiology | Year: 2014

Whole-genome sequencing (WGS) allows for effective tracing of Mycobacterium tuberculosis complex (MTBC) (tuberculosis pathogens) transmission. However, it is difficult to standardize and, therefore, is not yet employed for interlaboratory prospective surveillance. To allow its widespread application, solutions for data standardization and storage in an easily expandable database are urgently needed. To address this question, we developed a core genome multilocus sequence typing (cgMLST) scheme for clinical MTBC isolates using the Ridom SeqSphere+ software, which transfers the genome-wide single nucleotide polymorphism (SNP) diversity into an allele numbering system that is standardized, portable, and not computationally intensive. To test its performance, we performed WGS analysis of 26 isolates with identical IS6110 DNA fingerprints and spoligotyping patterns from a longitudinal outbreak in the federal state of Hamburg, Germany (notified between 2001 and 2010). The cgMLST approach (3,041 genes) discriminated the 26 strains with a resolution comparable to that of SNP-based WGS typing (one major cluster of 22 identical or closely related and four outlier isolates with at least 97 distinct SNPs or 63 allelic variants). Resulting tree topologies are highly congruent and grouped the isolates in both cases analogously. Our data show that SNP- and cgMLSTbased WGS analyses facilitate high-resolution discrimination of longitudinal MTBC outbreaks. cgMLST allows for a meaningful epidemiological interpretation of the WGS genotyping data. It enables standardized WGS genotyping for epidemiological investigations, e.g., on the regional public health office level, and the creation of web-accessible databases for global TB surveillance with an integrated early warning system. Copyright © 2014, American Society for Microbiology. All Rights Reserved.


Bos K.I.,University of Tübingen | Harkins K.M.,Arizona State University | Herbig A.,University of Tübingen | Coscolla M.,Swiss Tropical and Public Health Institute | And 30 more authors.
Nature | Year: 2014

Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those fromEurope, supporting the assumption that human tuberculosis was introduced post-contact1. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World2. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch3, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-oldmycobacterial genomesfromPeruvianhuman skeletons, revealing that amember of the M. tuberculosis complex caused human disease before contact.The ancient strains are distinct fromknownhuman-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean. © 2014 Macmillan Publishers Limited. All rights reserved.


Miotto P.,San Raffaele Scientific Institute | Cabibbe A.M.,San Raffaele Scientific Institute | Feuerriegel S.,Molecular Mycobacteriology | Feuerriegel S.,German Center for Infection Research | And 18 more authors.
mBio | Year: 2014

Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZAr). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZAr strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZAr strains, (iii) mutations with an unclear role found in less than 70% of PZAr strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZAr; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%.IMPORTANCE: Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients. © 2014 Miotto et al.


Roetzer A.,Molecular Mycobacteriology | Diel R.,University of Kiel | Kohl T.A.,Molecular Mycobacteriology | Kohl T.A.,Bielefeld University | And 10 more authors.
PLoS Medicine | Year: 2013

Background: Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains. Methods and Findings: During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed "Hamburg clone") started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance. Conclusions: Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context. Please see later in the article for the Editors' Summary. © 2013 Roetzer et al.


Niemann S.,Molecular Mycobacteriology | Niemann S.,German Center for Infection Research | Supply P.,French Institute of Health and Medical Research | Supply P.,French National Center for Scientific Research | And 2 more authors.
Cold Spring Harbor Perspectives in Medicine | Year: 2014

Genotyping of clinical Mycobacterium tuberculosis complex (MTBC) strains has become a standard tool for epidemiological tracing and for the investigation of the local and global strain population structure. Of special importance is the analysis of the expansion of multi- drug (MDR) and extensively drug-resistant (XDR) strains. Classical genotyping and, more recently, whole-genome sequencing have revealed that the strains of the MTBC are more diverse than previously anticipated. Globally, several phylogenetic lineages can be distin- guished whose geographical distribution is markedly variable. Strains of particular (sub)- lineages, such as Beijing, seem to be more virulent and associated with enhanced resistance levels and fitness, likely fueling their spread in certain world regions. The upcoming gener- alization of whole-genome sequencing approaches will expectedly provide more compre- hensive insights into the molecular and epidemiological mechanisms involved and lead to better diagnostic and therapeutic tools. © 2014 Cold Spring Harbor Laboratory Press. All rights reserved.


Weniger T.,University of Munster | Krawczyk J.,Molecular Mycobacteriology | Supply P.,Institute Pasteur Of Lille | Supply P.,French Institute of Health and Medical Research | And 3 more authors.
Infection, Genetics and Evolution | Year: 2012

Molecular diagnostics and genotyping of pathogens have become indispensable tools in clinical microbiology and disease surveillance. For isolates of the Mycobacterium tuberculosis complex (MTBC, causative agents of tuberculosis), multilocus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRU) has been internationally adopted as the new standard, portable, reproducible, and discriminatory typing method. Here, we review new sets of specialized web based bioinformatics tools that have become available for analyzing MLVA data especially in combination with other, complementary genotyping markers (polyphasic analysis). Currently, there are only two databases available that are not restricted to store one kind of genotyping data only, namely SITVIT/SpolDB4 and MIRU-VNTR. plus. SITVIT/SpolDB4 (http://www.pasteur-guadeloupe.fr:8081/SITVITDemo) contains spoligotyping data from a large number of strains of diverse origin. However, besides options to query the data, the actual version of SITVIT/SpolDB4 offers no functionality for more complex analysis e.g. tree-based analysis.In comparison, the MIRU-VNTR. plus web application (http://www.miru-vntrplus.org), represents a freely accessible service that enables users to analyze genotyping data of their strains alone or in comparison with a currently limited but well characterized reference database of strains representing the major MTBC lineages. Data (MLVA-, spoligotype-, large sequence polymorphism, and single nucleotide polymorphism) can be visualized and analyzed using just one genotyping method or a weighted combination of several markers. A variety of analysis tools are available such as creation of phylogenetic and minimum spanning trees, semi-automated phylogenetic lineage identification based on comparison with the reference database and mapping of geographic information. To facilitate scientific communication, a universal, expanding genotype nomenclature (MLVA MtbC15-9 type) service that can be queried via a web- or a SOAP-interface has been implemented. An extensive documentation guides users through all application functions. Perspectives for future development, including generalization to other bacterial species, are presented. © 2012 Elsevier B.V..


Feuerriegel S.,Molecular Mycobacteriology | Feuerriegel S.,German Center for Infection Research | Koser C.U.,University of Cambridge | Koser C.U.,Public Health England | And 2 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2014

Objectives: Sequence analysis of known antibiotic resistance genes of the Mycobacterium tuberculosis complex (MTBC) is increasingly being used to infer phenotypic resistance to a variety of antibiotics. However, a clear understanding of the genotype-phenotype relationship is required to interpret genotypic susceptibility results accurately. In this context, it is particularly important to distinguish phylogenetically informative neutral polymorphisms from true resistance-conferring mutations. Methods: Using a collection of 71 strains that encompasses all major MTBC genotypes, we mapped the genetic diversity in 18 genes that are known to be involved or were previously implicated in antibiotic resistance to eight current as well as two novel antibiotics. This included bedaquiline, capreomycin, ethambutol, fluoroquinolones, isoniazid, PA-824, para-aminosalicylic acid, prothionamide, rifampicin and streptomycin. Moreover, we included data from one of our prior studies that focused on two of the three known pyrazinamide resistance genes. Results: We found 58 phylogenetic polymorphisms that were markers for the genotypes M. tuberculosis Beijing, Haarlem, Latin American-Mediterranean (LAM), East African Indian (EAI), Delhi/Central Asian (CAS), Ghana, Turkey (Tur), Uganda I and II, Ural and X-type, as well as for Mycobacterium africanum genotypes West African I (WA I) and II (WA II), Mycobacterium bovis, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium microti and Mycobacterium canettii. Conclusions: This study represents one of the most extensive overviews of phylogenetically informative polymorphisms in known resistance genes to date, and will serve as a resource for the design and interpretation of genotypic susceptibility assays ©The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Weniger T.,University of Munster | Krawczyk J.,Molecular Mycobacteriology | Supply P.,University of Lille Nord de France | Niemann S.,Molecular Mycobacteriology | Harmsen D.,University of Munster
Nucleic Acids Research | Year: 2010

Harmonized typing of bacteria and easy identification of locally or internationally circulating clones are essential for epidemiological surveillance and disease control. For Mycobacterium tuberculosis complex (MTBC) species, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRU) has been internationally adopted as the new standard, portable, reproducible and discriminatory typing method. However, no specialized bioinformatics web tools are available for analysing MLVA data in combination with other, complementary typing data. Therefore, we have developed the web application MIRU-VNTRplus (http://www.miruvntrplus. org). This freely accessible service allows users to analyse genotyping data of their strains alone or in comparison with a reference database of strains representing the major MTBC lineages. Analysis and comparisons of genotypes can be based on MLVA-, spoligotype-, large sequence polymorphism and single nucleotide polymorphism data, or on a weighted combination of these markers. Tools for data exploration include search for similar strains, creation of phylogenetic and minimum spanning trees and mapping of geographic information. To facilitate scientific communication, an expanding genotype nomenclature (MLVA MtbC15-9 type) that can be queried via a web-or a SOAP-interface has been implemented. An extensive documentation guides users through all application functions. © The Author(s) 2010. Published by Oxford University Press.


Roetzer A.,Molecular Mycobacteriology | Schuback S.,Gesundheitsamt des Kreises Steinburg | Diel R.,Hannover Medical School | Gasau F.,Gesundheitsamt des Kreises Steinburg | And 5 more authors.
Journal of Clinical Microbiology | Year: 2011

In order to evaluate the discriminatory power of different methods for genotyping of Mycobacterium tuberculosis complex (MTBC) isolates, we compared the performance of (i) IS6110 DNA fingerprint typing, (ii) spoligotyping, and (iii) 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in a long-term study on the epidemiology of tuberculosis (TB) in Schleswig-Holstein, the northernmost federal state of Germany. In total, we analyzed 277 MTBC isolates collected from patients between the years 2006 and 2010. The collection comprised a broad spectrum of 13 different genotypes, among which strains of the Haarlem genotype (31%) were most prominent, followed by strains belonging to the Delhi and Beijing lineages (7% and 6%, respectively). On the basis of IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping analyses, 211 isolates had unique patterns (76%) and 66 isolates (24%) were in 20 clusters. MIRU-VNTR combined with spoligotyping analyses revealed 202 isolates with unique patterns (73%) and 75 isolates in 18 clusters (27%). Overall, there was 93.1% concordance between the typing results obtained; 198 strains were identified as unique, and 60 isolates were clustered by both typing combinations (including all 31 isolates with confirmed epidemiological links). Of the remaining 19 isolates with discrepant results, 15 were falsely clustered by MIRU-VNTR (six Beijing genotype strains) and four were clustered by IS6110 RFLP (low IS6110 copy number) only. In conclusion, in the study population investigated, a minority of isolates, especially of the Beijing genotype, clustered by standard 24-loci MIRU-VNTR and without an obvious epidemiological link may require second-line typing by IS6110 RFLP or hypervariable MIRU-VNTR loci. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

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