Sofía Samper

A new evolutionary scenario for the Mycobacterium tuberculosis complex

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. 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. 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.

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

BackgroundThe 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.ResultsThe 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.ConclusionOur 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.

An essential role for phoP in Mycobacterium tuberculosis virulence.

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.

Mutations in Putative Mutator Genes of Mycobacterium tuberculosis Strains of the W-Beijing Family

Alterations in genes involved in the repair of DNA mutations (mut genes) result in an increased mutation frequency and better adaptability of the bacterium to stressful conditions. W-Beijing genotype strains displayed unique missense alterations in three putative mut genes, including two of the mutT type (Rv3908 and mutT2) and ogt. These polymorphisms were found to be characteristic and unique to W-Beijing phylogenetic lineage. Analysis of the mut genes in 55 representative W-Beijing isolates suggests a sequential acquisition of the mutations, elucidating a plausible pathway of the molecular evolution of this clonal family. The acquisition of mut genes may explain in part the ability of the isolates of W-Beijing type to rapidly adapt to their environment.

IS6110 mediates increased transcription of the phoP virulence gene in a multidrug-resistant clinical isolate responsible for tuberculosis outbreaks.

ABSTRACT Drug resistance in Mycobacterium tuberculosis complex strains is solely due to chromosomal mutations that could affect bacterial virulence. Molecular epidemiology studies have shown that resistant strains are less likely to be clustered than susceptible strains. However, a few multidrug-resistant (MDR) M. tuberculosis complex strains have been described as causing outbreaks, suggesting that they have restored virulence or increased transmission. One of the biggest MDR tuberculosis outbreaks documented to date was caused by the B strain of M. bovis. Restriction fragment length polymorphism fingerprinting revealed that the B strain contains two copies of IS6110. Here, we mapped and sequenced the regions flanking the two copies of IS6110 in the B strain. Ligation-mediated PCR showed that one of these IS6110 copies is located within the promoter region of phoP, a transcriptional regulator that is essential for M. tuberculosis virulence. We used PCR to screen 219 MDR M. tuberculosis complex strains (90.4% of all MDR isolates) isolated in Spain between 1998 and 2002 and found that the B strain was the only strain that contained a copy of IS6110 in the phoP promoter. To determine whether IS6110 affects phoP promoter activity in the B strain, we individually cloned the phoP gene and its promoter region (including IS6110 from the B strain and the equivalent region from M. tuberculosis without IS6110 as a control) into a mycobacterial replicative plasmid and transformed M. smegmatis with the resulting plasmid. Primer extension analysis showed that phoP transcription was strongly upregulated when the promoter region contained IS6110, as in the case of the B strain.

Transmission between HIV-infected patients of multidrug-resistant tuberculosis caused by Mycobacterium bovis

Objective:To investigate outbreaks of multidrug-resistant tuberculosis (TB) by using DNA fingerprint databases. Design:Investigation of two outbreaks of multidrug-resistant TB in separate hospitals in Spain by restriction fragment length polymorphism (RFLP) and spoligotyping. Outbreak strains were compared with more than 1500 RFLPs of Mycobacterium tuberculosis complex strains isolated in Spain and 6000 RFLPs from 30 different countries. Methods:Standardized IS6110 DNA fingerprinting and ‘spoligotyping’ was used to type multidrug-resistant isolates belonging to the M. tuberculosis complex amongst the outbreak cases. The DNA types were matched against DNA fingerprint databases in Spain and The Netherlands. Results:The DNA typing analysis indicated that a single multidrug-resistant Mycobacterium bovisstrain was responsible for a nosocomial outbreak in a hospital in Spain involving at least 16 HIV-infected patients with non-treatable to multidrug-resistant TB. Introduction of the fingerprint type of this strain to the international database revealed a single matching strain. This strain was also isolated from an HIV-infected patient in The Netherlands who had died from multidrug-resistant TB. This patient had previously been hospitalized in Spain, where a multidrug-resistant TB nosocomial outbreak involving 20 HIV-infected patients was ongoing. The strains causing this outbreak were also identified as M. boviswith an identical DNA pattern to those strains isolated in the Spanish hospital and the patient in The Netherlands. Conclusions:The use of centralized DNA databases can help to identify rapidly the origin and transmission routes of multidrug-resistant TB across international boundaries and the potential use of such an early warning surveillance system for investigation of nosocomial multidrug-resistant TB outbreaks between HIV-infected patients. To our knowledge this is the first report of transmission of multidrug-resistant M. bovisbetween hospitals.

Proposal of a Consensus Set of Hypervariable Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat loci for Subtyping of Mycobacterium tuberculosis Beijing Isolates

ABSTRACT Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing.

Systematic Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Complex Isolates from Spain

ABSTRACT We used spoligotyping and restriction fragment length polymorphism (RFLP) of the IS6110-insertion sequence to study the molecular epidemiology of multidrug-resistant (MDR) tuberculosis in Spain. We analyzed 180 Mycobacterium tuberculosis complex isolates collected between January 1998 and December 2000. Consecutive isolates from the same patients (n = 23) always had identical genotypes, meaning that no cases of reinfection occurred. A total of 105 isolates (58.3%) had unique RFLP patterns, whereas 75 isolates (41.7%) were in 20 different RFLP clusters. Characterization of the katG and rpoB genes showed that 14 strains included in the RFLP clusters did not actually cluster. Only 33.8% of the strains isolated were suggestive of MDR transmission, a frequency lower than that for susceptible strains in Spain (46.6%). We found that the Beijing/W genotype, which is prevalent worldwide, was significantly associated with immigrants. The 22 isolates in the largest cluster corresponded to the Mycobacterium bovis strain responsible for two nosocomial MDR outbreaks in Spain.

Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela

BackgroundMolecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD).ResultsClinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised.Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively.Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis.ConclusionThis study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies.

GenoType MTBDRsl for Molecular Detection of Second-Line-Drug and Ethambutol Resistance in Mycobacterium tuberculosis Strains and Clinical Samples

ABSTRACT The purpose of this study was to evaluate the GenoType MTBDRsl assay (Hain Lifescience GmbH, Nehren, Germany) for its ability to detect resistance to fluoroquinolones (FLQ), injectable second-line antibiotics [kanamycin (KM) and capreomycin (CM)], and ethambutol (EMB) in Mycobacterium tuberculosis clinical strains and directly in clinical samples. A total of 34 clinical strains were characterized with the Bactec 460 TB system. Fifty-four clinical samples from 16 patients (5 were smear negative and 49 were smear positive) were also tested directly. The corresponding isolates of the clinical specimens were also analyzed with the Bactec 460TB. When there was a discrepancy between assays, pyrosequencing was performed. The overall rates of concordance of the MTBDRsl and the Bactec 460TB for the detection of FLQ, KM/CM, and EMB susceptibility in clinical strains were 72.4% (21/29), 88.8% (24/27), and 67.6% (23/34), whereas for clinical samples, rates were 86.5% (45/52), 92.3% (48/52), and 56% (28/50), respectively. In conclusion, the GenoType MTBDRsl assay may be a useful tool for making early decisions regarding KM/CM susceptibility and to a lesser extent regarding FLQ and EMB susceptibility. The test is able to detect mutations in both clinical strains and samples with a short turnaround time. However, for correct management of patients with extensively drug-resistant tuberculosis, results must be confirmed by a phenotypical method.