Rapid Differentiation of “
 Mycobacterium canettii
 ” from Other
 Mycobacterium tuberculosis
 Complex Organisms by PCR-Restriction Analysis of the
 hsp65
 Gene

Goh, Khye Seng; Legrand, Eric; Sola, Christophe; Rastogi, Nalin

doi:10.1128/jcm.39.10.3705-3708.2001

Cited by 37 publications

(44 citation statements)

References 17 publications

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“…When analyzing the polymorphism of the hsp65 gene by PCR restriction analysis (PRA) using HhaI, we found that 43 of the 44 smooth strains studied (including CIPT060) had the same specific "M. canettii" profile formerly described by Goh et al (8) and different from that of the MTBC strains. Figure 5A shows the constant pattern observed in representative strains from the MTBC.…”

Section: Resultsmentioning

confidence: 73%

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High Genetic Diversity Revealed by Variable-Number Tandem Repeat Genotyping and Analysis ofhsp65Gene Polymorphism in a Large Collection of “Mycobacterium canettii” Strains Indicates that theM. tuberculosisComplex Is a Recently Emerged Clone of “M. canettii”

et al. 2004

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We have analyzed, using complementary molecular methods, the diversity of 43 strains of "Mycobacterium canettii" originating from the Republic of Djibouti, on the Horn of Africa, from 1998 to 2003. Genotyping by multiple-locus variable-number tandem repeat analysis shows that all the strains belong to a single but very distant group when compared to strains of the Mycobacterium tuberculosis complex (MTBC). Thirty-one strains cluster into one large group with little variability and five strains form another group, whereas the other seven are more diverged. In total, 14 genotypes are observed. The DR locus analysis reveals additional variability, some strains being devoid of a direct repeat locus and others having unique spacers. The hsp65 gene polymorphism was investigated by restriction enzyme analysis and sequencing of PCR amplicons. Four new single nucleotide polymorphisms were discovered. One strain was characterized by three nucleotide changes in 441 bp, creating new restriction enzyme polymorphisms. As no sequence variability was found for hsp65 in the whole MTBC, and as a single point mutation separates M. tuberculosis from the closest "M. canettii" strains, this diversity within "M. canettii" subspecies strongly suggests that it is the most probable source species of the MTBC rather than just another branch of the MTBC.

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Section: Resultsmentioning

confidence: 73%

“…The "M. canettii" taxon can also be easily differentiated from the other members of the MTBC on the basis of a restriction site polymorphism in the hsp65 gene (8).…”

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confidence: 99%

High Genetic Diversity Revealed by Variable-Number Tandem Repeat Genotyping and Analysis ofhsp65Gene Polymorphism in a Large Collection of “Mycobacterium canettii” Strains Indicates that theM. tuberculosisComplex Is a Recently Emerged Clone of “M. canettii”

et al. 2004

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“…The restriction digest was visualized by agarose gel electrophoresis on 3% Metaphor (FMC Bioproducts, Rockland, Maine) and run in Tris-borate-EDTA (1ϫ) buffer, followed by ethidium-bromide staining. Pictures were taken electronically and were analyzed using the Taxotron software (PAD Grimont, Taxolab; Institut Pasteur) as previously reported (11).…”

Section: Methodsmentioning

confidence: 99%

Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase ( narGHJI ) Operon Promoter

et al. 2005

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A well-characterized collection of Mycobacterium tuberculosis complex (MTC) isolates, representing all known subspecies as well as some relevant genotypic families of M. tuberculosis, was analyzed for the newly discovered narGHJI ؊215 C-to-T promoter single-nucleotide polymorphism (SNP). This point mutation has been shown in earlier studies to be responsible for the differential nitrate reductase activity of M. tuberculosis versus M. bovis. As previously defined by the presence or the absence of the TbD1 genetic locus, the group included both the "modern" W-Beijing, Haarlem, and Central-Asian1 (CAS1) families as well as the "ancestral" East-African-Indian (EAI) clade. Interestingly, among "modern" M. tuberculosis isolates, those previously classified as Principal Genetic Group 1 (PGG1) organisms by katG 463 -gyrA 95 polymorphism analysis did not present the two-banded narGHJI restriction fragment length polymorphism analysis of PCR products pattern common to the other PGG1 MTC members, including the "ancestral" M. tuberculosis isolates. Instead, they showed a one-banded pattern, aligning them with other evolutionarily recent M. tuberculosis isolates of the PGG2 and PGG3 groups, such as Haarlem, Latin-American and Mediterranean (LAM), and X families. The presence of a nitrate reductase producer phenotype in "Mycobacterium canettii" and some "ancestral" M. tuberculosis isolates, despite a two-band ؊215C genotype, argues in favor of an alternate mechanism to explain the differential nitrate reductase activity of certain PGG1 subspecies of the MTC. Overall, these findings may help to establish the precise evolutionary history of important genotype families such as W-Beijing and suggest that the ؊215T genotype may have contributed the virulence, spread, and evolutionary success of "modern" M. tuberculosis strains compared to the remaining MTC organisms.Together with niacin production, catalase/peroxidase activity, and urease production, the assay of nitrate reductase activity, through the accumulation of nitrite, remains a basic phenotypic criterion for differentiating Mycobacterium tuberculosis from Mycobacterium bovis in diagnostic mycobacteriology (6). Contrary to M. tuberculosis, which presents a strong nitrate reductase activity under aerobic conditions, M. bovis and the M. bovis-derived BCG vaccine strain fail to rapidly accumulate nitrite from nitrate. However, both M. tuberculosis and M. bovis possess the narGHJI operon, which is expressed under anaerobic conditions in both subspecies and the product of which is a membrane-bound anaerobic nitrate reductase complex (25). As an alternative to using oxygen as a terminal electron acceptor, the NarGHJI complex couples this process to the reduction of nitrate. It was shown previously that this enzyme is induced under anaerobic conditions in Bacillus subtilis (18), and the switch to anaerobic metabolism is believed to be critical in the establishment of latent infection by M. tuberculosis and possibly the other tubercle bacilli. Recently, a C-to-T transition at posi...

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“…Once established in a clonal lineage, SNPs in the MTC organisms are believed to be relatively stable over time (56), and frequently identified SNPs in certain genes have been used, alone or in combination, as either specific markers for a given MTC species or to categorize isolates into defined MTC phylogenies (7,38,51). As examples, a unique signatory "M. canettii" SNP has been described in hsp65 (27), while commonly observed SNPs located in katG and gyrA are routinely assessed, because together they permit all MTC strains to be assigned into one of three principal genetic groups (PGG) (77). The distribution of the SNPs in katG and gyrA indicates that PGG1 strains are ancestral to PGG2 strains, which are in turn ancestral to PGG3 strains.…”

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confidence: 99%

Novel Genetic Polymorphisms That Further Delineate the Phylogeny of the Mycobacterium tuberculosis Complex

et al. 2006

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In a previous report, we described a PCR protocol for the differentiation of the various species of the Mycobacterium tuberculosis complex (MTC) on the basis of genomic deletions (R. C. Huard, L. C. de Oliveira Lazzarini, W. R. Butler, D. van Soolingen, and J. L. Ho, J. Clin. Microbiol. 41:1637-1650, 2003). That report also provided a broad cross-comparison of several previously identified, phylogenetically relevant, longsequence and single-nucleotide polymorphisms (LSPs and SNPs, respectively). In the present companion report, we expand upon the previous work (i) by continuing the evaluation of known MTC phylogenetic markers in a larger collection of tubercle bacilli (n ‫؍‬ 125), (ii) by evaluating additional recently reported MTC species-specific and interspecific polymorphisms, and (iii) by describing the identification and distribution of a number of novel LSPs and SNPs. Notably, new genomic deletions were found in various Mycobacterium tuberculosis strains, new species-specific SNPs were identified for "Mycobacterium canettii," Mycobacterium microti, and Mycobacterium pinnipedii, and, for the first time, intraspecific single-nucleotide DNA differences were discovered for the dassie bacillus, the oryx bacillus, and the two Mycobacterium africanum subtype I variants. Surprisingly, coincident polymorphisms linked one M. africanum subtype I genotype with the dassie bacillus and M. microti with M. pinnipedii, thereby suggesting closer evolutionary ties within each pair of species than had been previously thought. Overall, the presented data add to the genetic definitions of several MTC organisms as well as fine-tune current models for the evolutionary history of the MTC.Tuberculosis (TB) is an age-old, and now resurgent, infectious disease that has spread to nearly every corner of the globe. Mycobacteria that cause human and/or animal TB are grouped together within the Mycobacterium tuberculosis complex (MTC). The classical species of the MTC include Mycobacterium tuberculosis, Mycobacterium africanum (of various subtypes and variants), Mycobacterium microti, and Mycobacterium bovis (along with the widely used vaccine strain M. bovis bacillus Calmette-Guérin [BCG]) (7,38,51). Newly recognized additions to the MTC include Mycobacterium caprae and Mycobacterium pinnipedii (2, 12). Although it is not presently an officially described organism, "Mycobacterium canettii" is another widely accepted member of the MTC (85). Lastly, there exist rare MTC variants, the so-called dassie and oryx bacilli, whose standing within the MTC remains to be defined (50, 84). Historically, taxonomic segregation of the MTC has been based upon each species' unique combination of growth, morphological, physiological, and biochemical characteristics (57).Variations in geographical distribution, host preference, virulence, and relative human infectivity also differentiate the tubercle bacilli (38). Since M. tuberculosis sensu stricto is disproportionately responsible for the worldwide TB burden (38), it is of great interest to identify pote...

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Rapid Differentiation of “ Mycobacterium canettii ” from Other Mycobacterium tuberculosis Complex Organisms by PCR-Restriction Analysis of the hsp65 Gene

Cited by 37 publications

References 17 publications

High Genetic Diversity Revealed by Variable-Number Tandem Repeat Genotyping and Analysis ofhsp65Gene Polymorphism in a Large Collection of “Mycobacterium canettii” Strains Indicates that theM. tuberculosisComplex Is a Recently Emerged Clone of “M. canettii”

High Genetic Diversity Revealed by Variable-Number Tandem Repeat Genotyping and Analysis ofhsp65Gene Polymorphism in a Large Collection of “Mycobacterium canettii” Strains Indicates that theM. tuberculosisComplex Is a Recently Emerged Clone of “M. canettii”

Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase ( narGHJI ) Operon Promoter

Novel Genetic Polymorphisms That Further Delineate the Phylogeny of the Mycobacterium tuberculosis Complex

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