The distribution of 20 variable regions resulting from insertion-deletion 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. tuberculosi s 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. africanum→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.

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.

Two‐component regulatory proteins function in bacteria as sensory and adaptive factors in response to a wide range of environmental stimuli. Some two‐component systems, such as PhoP/PhoQ, control transcription of key virulence genes essential for survival in host cells in diverse intracellular bacterial pathogens, including Salmonella sp., Shigella sp. and Yersinia sp. In this study, we have disrupted the phoP gene from Mycobacterium tuberculosis, which codes for a putative transcription regulator factor of the two‐component system PhoP/PhoR. The phoP mutant strain exhibited impaired multiplication when cultured in mouse bone marrow‐derived macrophages. However, the mutation did not appear to affect survival of the organisms adversely inside macrophages. The mutant strain was also attenuated in vivo in a mouse infection model, with impaired growth observed in the lungs, livers and spleens. The results suggest that the phoP gene is required for intracellular growth of M. tuberculosis but is not essential for persistence of the bacilli.

Clinical antimicrobial susceptibility breakpoints are used to predict the clinical outcome of antimicrobial treatment. In contrast, microbiologic breakpoints are used to identify isolates that may be categorized as susceptible when applying clinical breakpoints but harbor resistance mechanisms that result in their reduced susceptibility to the agent being tested. Currently, the National Committee for Clinical Laboratory Standards (NCCLS) guidelines utilize clinical breakpoints to characterize the activity of the fluoroquinolones against Streptococcus pneumoniae. To determine whether levofloxacin breakpoints can identify isolates that harbor recognized resistance mechanisms, we examined 115 S. pneumoniae isolates with a levofloxacin MIC of >2 μg/mL for first-step parC mutations. A total of 48 (59%) of 82 isolates with a levofloxacin MIC of 2 μg/mL, a level considered susceptible by NCCLS criteria, had a first-step mutation in parC. Whether surveillance programs that use levofloxacin data can effectively detect emerging resistance and whether fluoroquinolones can effectively treat infections caused by such isolates should be evaluated.

Molecular epidemiological studies suggest that particular Mycobacterium tuberculosis strains have an enhanced capacity to spread within a community. One strain, the Beijing genotype, has been associated with outbreaks in a number of communities throughout the world. IS6110 restriction fragment length polymorphism (RFLP) analysis was performed on M. tuberculosis isolates from 566 of the 721 patients (78.5%) diagnosed with tuberculosis (TB) on Gran Canaria Island from 1993 to 1996, as well as 35% of isolates from 1991–1992 (85 strains). RFLP identification of the family of strains of the Beijing genotype was confirmed by spoligotyping. Medical records of all patients were reviewed and epidemiological links were identified. Of 566 M. tuberculosis isolates from 1993 to 1996 with RFLP available, 72% belonged to clusters. The largest contained 75 cases and was caused by a strain of the Beijing genotype that was introduced to the island in 1993. It was found in 10 patients in 1993 (5.5%), 12 in 1994 (8.1%), 18 in 1995 (16.4%), and 35 in 1996 (27.1%). Epidemiological linkage was confirmed for 68% of cases. This study has demonstrated rapid dissemination of this strain of the Beijing genotype. This genotype might play an important role in the future of the worldwide tuberculosis epidemic.