The distribution of 20 variable regions resulting from insertiondeletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and ''modern'' strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum3 M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.evolution ͉ diagnostic ͉ identification T he mycobacteria grouped in the Mycobacterium tuberculosis complex are characterized by 99.9% similarity at the nucleotide level and identical 16S rRNA sequences (1, 2) but differ widely in terms of their host tropisms, phenotypes, and pathogenicity. Assuming that they all are derived from a common ancestor, it is intriguing that some are exclusively human (M. tuberculosis, Mycobacterium africanum, Mycobacterium canettii) or rodent pathogens (Mycobacterium microti), whereas others have a wide host spectrum (Mycobacterium bovis). What was the genetic organization of the last common ancestor of the tubercle bacilli, and in which host did it live? Which genetic events may have contributed to the fact that the host spectrum is so different and often specific? Where and when did M. tuberculosis evolve? Answers to these questions are important for a better understanding of the pathogenicity and the global epidemiology of tuberculosis and may help to anticipate future trends in the spread of the disease.Because of the unusually high degree of conservation in their housekeeping genes, it has been suggested that the members of the M. tuberculosis complex underwent an evolutionary bottleneck at the time of speciation, estimated to have occurred roughly 15,000-20,000 years ago (2). Also, it has been speculated tha...

Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock-in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1-encoded proteins were localized in the cell wall, and two of them, the immunodominant T-cell antigens ESAT-6 and CFP-10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock-ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock-in, whereas BCG controls were cleared. Knocking-in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.

The addition of bedaquiline to a preferred background regimen for 24 weeks resulted in faster culture conversion and significantly more culture conversions at 120 weeks, as compared with placebo. There were more deaths in the bedaquiline group than in the placebo group. (Funded by Janssen Pharmaceuticals; TMC207-C208 ClinicalTrials.gov number, NCT00449644.).

The clinical activity of TMC207 validates ATP synthase as a viable target for the treatment of tuberculosis. (ClinicalTrials.gov number, NCT00449644.)

The live tuberculosis vaccines Mycobacterium bovis BCG (bacille Calmette-Guérin) and Mycobacterium microti both lack the potent, secreted T-cell antigens ESAT-6 (6-kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein). This is a result of independent deletions in the region of deletion-1 (RD1) locus, which is intact in virulent members of the Mycobacterium tuberculosis complex. To increase their immunogenicity and protective capacity, we complemented both vaccines with different constructs containing the esxA and esxB genes, which encode ESAT-6 and CFP-10 respectively, as well as a variable number of flanking genes. Only reintroduction of the complete locus, comprising at least 11 genes, led to full secretion of the antigens and resulted in specific ESAT-6-dependent immune responses; this suggests that the flanking genes encode a secretory apparatus. Mice and guinea pigs vaccinated with the recombinant strain BCG::RD1-2F9 were better protected against challenge with M. tuberculosis, showing less severe pathology and reduced dissemination of the pathogen, as compared with control animals immunized with BCG alone.

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BackgroundThe continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents.Methods and FindingsWe performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T [isoniazid]; gidB 130 bp deletion [streptomycin]; 1957 [95% highest posterior density (HPD): 1937–1971]), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P [rifampicin]; pncA 1 bp insertion [pyrazinamide]; 1984 [95% HPD: 1974–1992]) and 10 y (rpoB D435G [rifampicin]; rrs 1400 [kanamycin]; gyrA A90V [ofloxacin]; 1995 [95% HPD: 1988–1999]) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe.ConclusionsIn the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic e...

Beijing/W strains of Mycobacterium tuberculosis are geographically widespread and hypervirulent. To enhance our understanding of their origin and evolution, we sought phylogenetically informative large sequence polymorphisms (LSPs) within the Beijing/W family. Comparative whole-genome hybridization of Beijing/W strains revealed 21 LSPs, 7 of which were previously unreported. We show that some of these LSPs are unique event polymorphisms that can be used to define and subdivide the Beijing/W family. One LSP (RD105) was seen in all Beijing/W strains and thus serves as a useful marker for the identification of this family of strains. Additional LSPs (RD142, RD150, and RD181) further divided this family into four monophyletic subgroups, demonstrating a deeper population structure than previously appreciated. All Beijing/W strains were also observed to have an intact pks15/1 gene that is involved in the biosynthesis of a phenolic glycolipid, a putative virulence factor. A simple PCR assay using these Beijing/W strain-defining deletions will facilitate molecular epidemiological studies and may assist in the identification of the molecular basis of phenotypes associated with this important lineage of M. tuberculosis.Molecular epidemiological studies have identified a genetically related group of Mycobacterium tuberculosis strains called Beijing/W that are widespread in many regions of the world (3, 10). Strains from this group have been associated with drug resistance, most notably in an outbreak of multidrug-resistant tuberculosis in New York (1, 4, 5, 35) and more recently in Russian prisons (31,32). This association with drug resistance led to the hypothesis that strains from the Beijing/W group might be hypermutable (25), but this has recently been refuted (37). In Vietnam, Beijing/W strains were also associated with relapses and treatment failures (17). In the laboratory, these strains have a hypervirulent phenotype in mice and elicit a distinct immune response with a reduced level of cytokines, resulting in a failure to induce Th1 type immunity (19,20) of which a polyketide synthase-derived phenolic glycolipid (PGL) is thought to play a principal role (26). Beijing/W strains also have a faster growth rate within human monocytes than do control strains (18). The bacterial genetic factors underlying this virulence are unknown, but the genetic relatedness of these Beijing/W strains suggests clonal expansion (19,25,29). It has even been hypothesized that vaccination with Mycobacterium bovis BCG may protect less well against Beijing/W strains, suggesting that they are escape variants (11,19,36).Beijing/W strains share genetic markers, such as similar IS6110 restriction fragment length polymorphism patterns and spoligotypes (3, 15). Currently, Beijing/W strains are principally identified by the number of spacers in the direct repeat (DR) region of the M. tuberculosis genome, namely, spoligotype S00034, or ST1, which is characterized by the deletion of spacers 1 to 34 (36) and, in a global survey of spoligotypes, type S...