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

Abstract

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...

Antemortem Diagnosis of Mycobacterium bovis Infection in Free-ranging African Lions (Panthera leo) and Implications for Transmission

“…The remaining pellets were suspended in 1 mL PBS, and 500 mL of the suspension was inoculated into a Mycobacteria Growth Indicator Tube (MGIT) and incubated in a BACTEC TM MGIT 960 Mycobacterial Detection System (both Becton Dickinson). Cultures that were Ziehl-Neelsen stain-positive were identified to species by sequencing fragments of the 16S ribosomal DNA (Harmsen et al 2003) and gyrB genes (Huard et al 2006).…”

Rapid detection of Mycobacterium tuberculosis complex in cattle and lechwe (Kobus leche kafuensis) at the slaughter house

“…M. bovis was identified from the results of the multiplex-PCR, which targets three genetic regions that are cfp32, RD9 and RD12. 6,[22][23][24] In summary, our study clearly provides evidence that the LAMP assay is a method that allows direct detection of MTC from processed clinical/abattoir specimens. This means that the technique can also be applied in the field of veterinary medicine for rapid confirmation of suspected cases.…”

“…These include M. tuberculosis, M. africanum, M. microti, M. bovis, M. caprae, and M. pinnipedii. [4][5][6] Some of these Mycobacterium species are major reemerging zoonotic agents of bovine TB, the prevalence of which depends on direct exposure to cattle and consumption of unpasteurized dairy products. 7,8 Furthermore, the interaction in the human-livestock-wildlife interphase areas of some wildlife animals like lechwe (Kobus leche kafuensis), documented with M. bovis, have broadened the reservoir base for MTC.…”

Molecular characterization of Mycobacterium tuberculosis isolates from elephants of Nepal

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“…20 The spoligotype was determined as previously described. 21 Briefly, the direct-repeat (DR) region was amplified with a primer pair and the PCR products were hybridized to a set of 43 oligonucleotide probes corresponding to each spacer, which were covalently bound to the membrane.…”