The highly successful human pathogen Mycobacterium tuberculosis has an extremely low level of genetic variation, which suggests that the entire population resulted from clonal expansion following an evolutionary bottleneck around 35,000 y ago. Here, we show that this population constitutes just the visible tip of a much broader progenitor species, whose extant representatives are human isolates of tubercle bacilli from East Africa. In these isolates, we detected incongruence among gene phylogenies as well as mosaic gene sequences, whose individual elements are retrieved in classical M. tuberculosis. Therefore, despite its apparent homogeneity, the M. tuberculosis genome appears to be a composite assembly resulting from horizontal gene transfer events predating clonal expansion. The amount of synonymous nucleotide variation in housekeeping genes suggests that tubercle bacilli were contemporaneous with early hominids in East Africa, and have thus been coevolving with their human host much longer than previously thought. These results open novel perspectives for unraveling the molecular bases of M. tuberculosis evolutionary success.

Global spread and genetic monomorphism are hallmarks of Mycobacterium tuberculosis, the agent of human tuberculosis. In contrast, Mycobacterium canettii, and related tubercle bacilli that also cause human tuberculosis and exhibit unusual smooth colony morphology, are restricted to East-Africa. Here, we sequenced and analyzed the genomes of five representative strains of smooth tubercle bacilli (STB) using Sanger (4-5x coverage), 454/Roche (13-18x coverage) and/or Illumina DNA sequencing (45-105x coverage). We show that STB are highly recombinogenic and evolutionary early-branching, with larger genome sizes, 25-fold more SNPs, fewer molecular scars and distinct CRISPR-Cas systems relative to M. tuberculosis. Despite the differences, all tuberculosis-causing mycobacteria share a highly conserved core genome. Mouse-infection experiments revealed that STB are less persistent and virulent than M. tuberculosis. We conclude that M. tuberculosis emerged from an ancestral, STB-like pool of mycobacteria by gain of persistence and virulence mechanisms and we provide genome-wide insights into the molecular events involved.

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

We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.

We analyzed DNA polymorphisms in 455 Mycobacterium tuberculosis complex isolates from 455 patients to evaluate the biodiversity of tubercle bacilli in Ouest province, Cameroon. The phenotypic and genotypic identification methods gave concordant results for 99.5% of M. tuberculosis isolates (413 strains) and for 90% of Mycobacterium africanum isolates (41 strains). Mycobacterium bovis was isolated from only one patient. Analysis of regions of difference (RD4, RD9, and RD10) proved to be an accurate and rapid method of distinguishing between unusual members of the M. tuberculosis complex. Whereas M. africanum strains were the etiologic agent of tuberculosis in 56% of cases 3 decades ago, our results showed that these strains now account for just 9% of cases of tuberculosis. We identified a group of closely genetically related M. tuberculosis strains that are currently responsible for >40% of smear-positive pulmonary tuberculosis cases in this region of Cameroon. These strains shared a spoligotype lacking spacers 23, 24, and 25 and had highly related IS6110 ligation-mediated (LM) PCR patterns. They were designated the "Cameroon family." We did not find any significant association between tuberculosis-causing species or strain families and patient characteristics (sex, age, and human immunodeficiency virus status). A comparison of the spoligotypes of the Cameroon strains with an international spoligotype database (SpolDB3) containing 11,708 patterns from >90 countries, showed that the predominant spoligotype in Cameroon was limited to West African countries (Benin, Senegal, and Ivory Coast) and to the Caribbean area.In 1993, the World Health Organization declared tuberculosis (TB) a global emergency. One-third of the world's population is infected by Mycobacterium tuberculosis complex strains, the etiologic agents of TB. Although Ͻ10% of infected people actually develop active TB during their lifetimes, this represents 8 million new cases of TB each year, including 3.5 million (44%) cases of smear-positive pulmonary disease, leading to 1.9 million deaths per year (5, 6). Ninety-five percent of cases occur in developing countries, where the lack of proper health care systems leads to incomplete case and contact tracing, incomplete treatment, and an increase in drug resistance. Due to the powerful interaction between TB and human immunodeficiency virus (HIV) disease, together with the problems of poverty and malnutrition, the incidence of TB is increasing dramatically in sub-Saharan Africa (22).In Cameroon, a country with 15 million inhabitants, the incidence of TB in 2000 was estimated at Ͼ300 cases per 100,000 inhabitants in the last World Health Organization report (29), with an estimated 21,594 new sputum smear-positive cases. Although there is a paucity of information regarding the distribution of M. tuberculosis complex strains in Cameroon, one study performed 30 years ago (14) reported that 56% of cases of TB were due to Mycobacterium africanum strains in Ouest and Sud provinces, Cameroon.Several intervention s...

Molecular epidemiologic findings suggest an ancient focus of TB.

In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. (J. Clin. Microbiol. 37:2607-2618, 1999). This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future.

A preliminary investigation of the genetic biodiversity of Mycobacterium tuberculosis complex strains in Cameroon, a country with a high prevalence of tuberculosis, described a group of closely related M. tuberculosis strains (the Cameroon family) currently responsible for more than 40% of smear-positive pulmonary tuberculosis cases. Here, we used various molecular methods to study the genetic characteristics of this family of strains. Cameroon family M. tuberculosis strains (i) are part of the major genetic group 2 and lack the TbD1 region like other families of epidemic strains, (ii) lack spacers 23, 24, and 25 in their direct repeat ( Molecular epidemiology methods revolutionized the fields of research, prevention, and control of tuberculosis (TB), allowing the differentiation between strains, assessment of the overall diversity of Mycobacterium tuberculosis complex strains including differences by region and population, and measurement of the prevalence of endemic strains (28). However, few molecular epidemiological studies have been conducted in countries with a high incidence of TB. The available data suggest that families of closely related strains are common in these areas (12). The "Beijing family" is one of the most well-known families, highly prevalent in East Asia and widespread around the world (11).Molecular analysis based on several variable genomic regions is required for a good definition of strains belonging to different families. Restriction fragment length polymorphism (RFLP) analysis based on the insertion sequence IS6110 results in a unique genotype since both the number of copies of this genetic element and its positions in the genome are variable (25,26). Precise IS6110 insertion site mapping provides additional information on the fitness of the strain (1) given that IS6110 insertion can modify the expression of the gene involved. Another genetic element useful for characterizing tubercle bacilli is the direct repeat (DR) locus (13), a polymorphic insertion preferential locus (ipl) for IS6110. DR polymorphism can be analyzed by spoligotyping, a method involving PCR-reverse hybridization (14). The DR locus is likely to evolve more slowly than IS6110, making spoligotyping less adequate than IS6110-RFLP for discriminating strains but more convenient for investigating the biogeographic distribution of families of M. tuberculosis complex strains (32). Variable-number tandem repeats, named mycobacterial interspersed repetitive units (MIRU-VNTR), are another type of variable element in the M. tuberculosis complex genome showing extensive polymorphism (17) with a discrimination power close to that of IS6110-RFLP (6). Because of their stability, they can be used for a clear definition of families of tubercle bacilli as well.Strains of the M. tuberculosis complex exhibit very little genome sequence diversity except in repeat sequences. Consequently, insignificant and rare genome alterations inherited and maintained through long-term evolution can be of phylogenetic value. On the basis of polymorphi...