Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology.

BACKGROUND: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database. RESULTS: The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network. CONCLUSION: Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

Genotyping of Mycobacterium tuberculosis clinical isolates in two cities of Turkey: description of a new family of genotypes that is phylogeographically specific for Asia Minor.

BACKGROUND: Population-based bacterial genetics using repeated DNA loci is an efficient approach to study the biodiversity and phylogeographical structure of human pathogens, such as Mycobacterium tuberculosis, the agent of tuberculosis. Indeed large genetic diversity databases are available for this pathogen and are regularly updated. No population-based polymorphism data were yet available for M. tuberculosis in Turkey, at the crossroads of Eurasia. RESULTS: A total of 245 DNAs from Mycobacterium tuberculosis clinical isolates from tuberculosis patients residing in Turkey (Malatya n = 147 or Ankara n = 98) were genotyped by spoligotyping, a high-throughput genotyping method based on the polymorphism of the Direct Repeat locus. Thirty-three spoligotyping-defined clusters including 206 patients and 39 unique patterns were found. The ST41 cluster, as designated according to the international SpolDB3 database project, represented one fourth and when gathered to three genotypes, ST53, ST50 and ST284, one half of all the isolates. Out of 34 clinical isolates harboring ST41 which were further genotyped by IS6110 and by MIRU-VNTR typing, a typical 2-copy IS6110-RFLP pattern and a "215125113322" MIRU-VNTR pattern were observed among 21 clinical isolates. Further search in various databases confirms the likely Turkish-phylogeographical specificity of this clonal complex. CONCLUSION: We described a new phylogeographically-specific clone of M. tuberculosis, designated LAM7-TUR. Further investigations to assess its frequency within all regions of Turkey and its phylogeographical origin and phylogenetic position within the global M. tuberculosis phylogenetic tree will shed new light on its endemicity in Asia Minor.

Utility of fast mycobacterial interspersed repetitive unit-variable number tandem repeat genotyping in clinical mycobacteriological analysis.

BACKGROUND: Analysis of variable numbers of tandem repeats (VNTR) of genetic elements called mycobacterial interspersed repetitive units (MIRUs) is a recently described, polymerase chain reaction (PCR)-based method used to genotype Mycobacterium tuberculosis. It is much faster, requires a smaller amount of DNA, and has approximately the same discriminatory power as the standard IS6110 restriction fragment-length polymorphism (RFLP) method. We report the adaptation and optimization of MIRU-VNTR genotyping on a capillary electrophoresis system. We describe its application to 3 typical clinical situations encountered in our laboratory (Institut Pasteur de Bruxelles, Laboratoire Tuberculose et Mycobacteries; Brussels, Belgium). METHODS: MIRU-VNTR genotyping was performed on heat-inactivated M. tuberculosis cultures obtained from clinical specimens on Lowenstein solid medium or in mycobacteria growth indicator liquid tubes (Becton Dickinson). After amplification of 12 genomic loci using 4 different multiplex PCRs, DNA fragments were separated by capillary electrophoresis using the ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems). Sizing of the PCR fragments and assignment of the various MIRU-VNTR alleles were done using the GeneScan and customized Genotyper software packages (PE Applied Biosystem). RESULTS: Clustering on the basis of IS6110 fingerprinting of isolates from 3 different patients attending the same hospital was confirmed by MIRU-VNTR typing. This concordance between 2 independent, highly discriminatory techniques was decisive in triggering an epidemiological inquiry that led to identification of a bronchoscopy-related tuberculosis nosocomial infection. A mixed tuberculosis infection in a patient whose infection was initially suspected as a result of the IS6110 RFLP method was clearly identified by MIRU-VNTR typing. Finally, automated MIRU-VNTR analysis permitted the identification of laboratory contamination in 6 liquid cultures of M. tuberculosis within several hours. CONCLUSION: These examples illustrate the utility of this genotyping technique for quick and accurate resolution of problems commonly encountered in clinical mycobacteriology.

Population-based molecular epidemiological study of tuberculosis in Malatya, Turkey.

This investigation describes drug resistance patterns and genotyping data on a total of 145 Mycobacterium tuberculosis strains isolated between 2000 and 2004 in Malatya, Turkey. Drug susceptibility results indicated a total of 20% resistant and 4.8% of multidrug-resistant isolates. Spoligotyping resulted in 25 unique patterns and 120 strains in 19 clusters (2 to 33 strains per cluster). When the results were compared to an international spoligotyping database, 19 of 25 unique patterns matched existing shared spoligotype international types (SITs). This led to the description of 38 SITs with 139 strains and 6 orphan patterns (not previously reported). Five of the SITs (SIT759, SIT1936, SIT1937, SIT1938, and SIT2285) were newly created. The most prevalent spoligotype was SIT41 (LAM7-TUR) with 33 (23.9%) isolates. The repartition of strains according to major M. tuberculosis clades (in decreasing order) was as follows: ill-defined T clade (45.7%) > Latin American and Mediterranean (LAM; 29%) > Haarlem (15.9%). Strains belonging to Central Asian (CAS), East-African Indian (EAI), Beijing, and Africanum clades were absent in this setting. IS6110-restriction fragment length polymorphism (RFLP) resulted in 19 clusters (52 strains), with a final clustering rate of 35.9% and a recent transmission rate of 22.8%. Typing based on mycobacterial interspersed repetitive units (MIRUs) permitted us to identify 65 patterns (23 orphan patterns and 42 patterns that matched existing MIRU international types in an updated database). The combination of the three typing methods allowed us to calculate a final clustering rate of 22% and a significantly lower transmission rate of 13.1%. The discrimination achieved by IS6110-RFLP/MIRUs was not significantly improved by adding spoligotyping results (1.4%). We conclude that our patient population is infected by diverse M. tuberculosis populations; however, the majority of the ongoing transmission is due to "evolutionary recent" tuberculosis lineages belonging to principal genetic group 2 (PGG2; Haarlem and LAM) and PGG3 (ill-defined T clade), and most of it is attributable to the LAM7-TUR sublineage with an enhanced phylogeographical specificity for Turkey. An absence of lineages belonging to PGG1 clones (EAI, CAS, and Beijing, essentially found in Central, South, and Southeast Asia), is noteworthy.

Evaluation of the epidemiological relevance of variable-number tandem-repeat genotyping of Mycobacterium bovis and comparison of the method with IS6110 restriction fragment length polymorphism analysis and spoligotyping.

Sources of Mycobacterium bovis contamination remain unclear for many cases of animal and human disease. A major limitation is the lack of sufficiently informative or epidemiologically well evaluated molecular methods for typing. Here, we report an evaluation of a high-throughput method based on 29 mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) loci to genotype 127 M. bovis isolates from cattle from 77 different Belgian farms, representative of a nationwide collection obtained from 1995 to 2003. MIRU-VNTR stability was demonstrated by analyzing a series of 74 isolates in total, obtained from different animals from a single farm or from different farms with an identified epidemiological link. The genotyping results and the genotypic diversity (h) were compared with those obtained by IS6110 restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Among 68 isolates with no known epidemiological link, MIRU-VNTR typing discriminated better than either RFLP analysis or spoligotyping, [corrected] taken individually (32 versus 16 and 17 genotypes; h = 0.91 versus 0.73 and 0.85, respectively) or in combination (32 versus 28 genotypes; h = 0.91 versus 0.92). Maximal resolution was already achieved with a subset of 9 loci. The observed congruence of the genetic relationships based on IS6110 RFLP analysis, spoligotyping, and MIRU-VNTR markers is consistent with a clonal population structure of M. bovis. These results support MIRU-VNTR typing as a convenient and discriminatory technique for analysis of the population structure of M. bovis in much greater detail and for addressing some still unresolved issues in the epidemiology of the pathogen.

Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis.

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%--and by 23% in combination with spoligotyping--among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold--by threefold in combination with spoligotyping--under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.

The protein kinase kin1 is required for cellular symmetry in fission yeast.

The fission yeast Schizosaccharomyces pombe is a highly polarized unicellular eukaryote with two opposite growing poles in which F-actin cytoskeleton is focused. The KIN1/PAR-1/MARK protein family is composed of conserved eukaryotic serine/threonine kinases which are involved in cell polarity, microtubule stability or cell cycle regulation. Here, we investigate the function of the fission yeast KIN1/PAR-1/MARK member, kin1p. Using a deletion allele (kin1Delta), we show that kin1 mutation promotes a delay in septation. Kin1p regulates the structure of the new cell end after cytokinesis by modulating cell wall remodeling. Abnormal shaped interphase kin1Delta cells misplace F-actin patches and the premitotic nucleus. Thus, mitotic kin1Delta cells misposition the F-actin ring assembly site that is dependent on the position of the interphase nucleus. The resulting asymmetric cell division produces daughter cells with distinct shapes. Overexpressed kin1p accumulates asymmetrically at the cell cortex and affects cell shape, F-actin organization and microtubules. Our results suggest that correct dosage of kin1p at the cortex is required for spatial organization of the fission yeast cell.