Igor Mokrousov

Bio: Igor Mokrousov is an academic researcher from Pasteur Institute. The author has contributed to research in topics: Mycobacterium tuberculosis & Tuberculosis. The author has an hindex of 41, co-authored 142 publications receiving 6332 citations. Previous affiliations of Igor Mokrousov include Capital Medical University.

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

Abstract: 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.

Evolutionary history and global spread of the Mycobacterium tuberculosis Beijing lineage

Abstract: Mycobacterium tuberculosis strains of the Beijing lineage are globally distributed and are associated with the massive spread of multidrug-resistant (MDR) tuberculosis in Eurasia. Here we reconstructed the biogeographical structure and evolutionary history of this lineage by genetic analysis of 4,987 isolates from 99 countries and whole-genome sequencing of 110 representative isolates. We show that this lineage initially originated in the Far East, from where it radiated worldwide in several waves. We detected successive increases in population size for this pathogen over the last 200 years, practically coinciding with the Industrial Revolution, the First World War and HIV epidemics. Two MDR clones of this lineage started to spread throughout central Asia and Russia concomitantly with the collapse of the public health system in the former Soviet Union. Mutations identified in genes putatively under positive selection and associated with virulence might have favored the expansion of the most successful branches of the lineage.

Snapshot of Moving and Expanding Clones of Mycobacterium tuberculosis and Their Global Distribution Assessed by Spoligotyping in an International Study

Abstract: The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.

Global distribution of Mycobacterium tuberculosis spoligotypes.

Abstract: Since the publication of the second version of our spoligotypes database on Mycobacterium tuberculosis (1), the causative agent of tuberculosis (TB), the proportion of clustered isolates (shared types [STs]) increased from 84% (2,779/3,319) to 90% (11,708/13,008). Fifty percent of the clustered isolates were found in only 20 STs. Three of these isolates are M. bovis, including M. bovis BCG (ST 481, 482, and 683). The addition of the next 30 most frequent STs increased the total proportion of clustered isolates (65% instead of 50% initially). A total of 36 potential subfamilies or subclades of M. tuberculosis complex have been tentatively identified, leading to the definition of major and minor visual recognition rules (Table). The ancestral East-African Indian family (EAI) is made up of at least five main subclades, whereas at least three major spoligotyping patterns are found within the Haarlem family (2). Two families found in central and Middle Eastern Asia (CAS1 and CAS2) are newly defined. The X family (3) is also currently split into at least three well-defined subclades. However, the subdivision of family T (T1–T4, likely to represent relatively old genotypes), which differs from the classic ST 53 (all spacers present except 33–36), remains poorly defined. Similarly, the Latino-American and Mediterranean family (LAM) is tentatively split into subclades LAM1–LAM10 (4). Spoligotyping used alone is not well suited for studying the phylogeny of these two clades (T and LAM). Such study will require results from other genotyping methods such as IS6110-restriction fragment length polymorphism (5) or mycobacterial interspersed repetitive units–variable number of DNA tandem repeats (6). Among well-characterized major clades of tubercle bacilli, four families represent 35% of 11,708 clustered isolates (Beijing 11%, LAM 9.3%, Haarlem 7.5%, and the X clade 7%). Table Excerpt from SpolDB3 database showing prototype spoligotypes, visual recognition rules, and binary and octal descriptiona The global distribution of the most frequently observed spoligotypes by continent in SpolDB3 is as follows. Among the patterns originating in North America (n= 4,276, 32% of the total number of isolates in the database), 16% of the strains are of the Beijing type, 14% belong to ST 137 or ST 119 (X family), and 8% are unique (results not shown). In Central America (n=587, 4.5%), 8% of the strains belong to the ubiquitous ST 53, 7% are ST 50, and 6% are ST 2; the last two STs are part of the Haarlem family. In South America (n=861, 6.6%), the distribution of ST 53 and ST 50 accounts for 10% and 9%, respectively, of the spoligotypes, whereas ST 42 accounts for as much as 9% of the total isolates. The origin of ST 42 remains to be established. In Africa (n=1,432, 11%), ST 59 and ST 53 account for 9% of all isolates studied thus far; however, the values obtained for ST 59 are biased because strains from Zimbabwe are overrepresented. We also observed that M. africanum ST 181 accounts for as much as 6% of all spoligotypes from Africa in our sample. In Europe (n=4,360, 33.5%), ST 53 represents as much as 9% of the spoligotypes, ST 50 and 47 (Haarlem family) represent 8% of the cases, and the Beijing family accounts for 4% of the spoligotypes. In the Middle Eastern and central Asian region, where the number of samples obtained is still very low (n=351, 2.7%), a high diversity of strains within the EAI and CAS families has been observed, and no single pattern currently exceeds 5%. Further studies of isolates from these regions are needed, e.g., in India, where our sampling is still anecdotal (n=44 isolates). Notwithstanding the scarcity of available data from this region, the observed diversity suggests that this region might be of great interest for further study of the genetic variation of tubercle bacilli. Contrary to what we observed for the Middle East and central Asia, the Far East Asian region (n=801, 6.1%) is characterized by the prevalence of a single genotype, the Beijing type family, a family linked to emerging multiresistance (7). One out of two strains in the Far East is a Beijing type. In Oceania (n=340, 2.6%), ST 19 and Beijing account for 15% and 13%, respectively, of clustered isolates. Thus, this preliminary analysis of the spoligotype distribution of SpolDB3 clearly shows major differences in the population structure of tubercle bacilli within the eight subcontinents studied (Africa; Europe; North America; Central America; South America; Middle East and Central Asia; Far East Asia; and Oceania). At present, SpolDB 3 is an experimental tool that has yet to prove its usefulness in tracking epidemics. Nevertheless, the facility with which matches between spoligotypes can be detected suggests that this tool may be a good screening mechanism for population-based studies on recent TB transmission. Indeed, the detection of a rarely found ST in SpolDB3 may be a catalyst that signals researchers to look for the clonality of the isolates and to study their epidemiologic relatedness. Data-exchange protocols through inter-networking will also be implemented in the near future. Working groups such as the European Network for Exchange of Molecular Typing Information (available from: URL: www.rivm.nl/enemti) are coordinating such initiatives. The expanded use of the Bionumerics software (third upgrade; Applied Maths, St. Martens-Latem, Belgium) may also foster this research field. SpolDB3 will also be instrumental in facilitating better understanding of the driving forces that shape tubercle bacilli evolution. Further research should now emphasize the use of data-mining methods, in combination with experts’ knowledge, to tackle the complex dynamics of the population's genetics of tubercle bacilli and TB transmission (3). Our sample represents the compilation of many national studies and, as such, should be considered as an ongoing population-based project aimed at studying global TB genetic diversity. Nevertheless, obtaining a more precise and representative snapshot of the genetic variability of M. tuberculosis complex will require a larger sampling. Although only partially representative of worldwide spoligotypes of M. tuberculosis complex, Spo1DB3 contains a reservoir of genetic information that has already proved useful for defining the phylogenetic links that exist within the TB genomes and for constructing theoretical models of genome evolution. Much remains to be done to evaluate the potential of global genetic databases to better characterize casual contacts (that could lead to identification of sporadic cases) in TB epidemiology. An improved version of our database, which will focus on areas with a high prevalence of TB, is currently in development; as of August 26, 2002, it had 20,000 isolates and 3,000 alleles. Ongoing population-based genotyping projects will likely help shed light on contemporary and ancient tubercle bacilli’s evolutionary history.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

Abstract: The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

RNA-guided genetic silencing systems in bacteria and archaea

Abstract: Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.

CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats.

Abstract: Clustered regularly interspaced short palindromic repeats (CRISPRs) constitute a particular family of tandem repeats found in a wide range of prokaryotic genomes (half of eubacteria and almost all archaea). They consist of a succession of highly conserved regions (DR) varying in size from 23 to 47 bp, separated by similarly sized unique sequences (spacer) of usually viral origin. A CRISPR cluster is flanked on one side by an AT-rich sequence called the leader and assumed to be a transcriptional promoter. Recent studies suggest that this structure represents a putative RNA-interference-based immune system. Here we describe CRISPRFinder, a web service offering tools to (i) detect CRISPRs including the shortest ones (one or two motifs); (ii) define DRs and extract spacers; (iii) get the flanking sequences to determine the leader; (iv) blast spacers against Genbank database and (v) check if the DR is found elsewhere in prokaryotic sequenced genomes. CRISPRFinder is freely accessible at http://crispr.u-psud.fr/Server/CRISPRfinder.php.

Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis

Abstract: 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.