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, long-sequence 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.

FIG. 1.

Composite revised MTC PCR typing panel. Illustrated is an example of the MTC PCR typing panel output pattern for a single M. tuberculosis isolate (strain H37Ra). The protocol was amended from reference to include an amplification test for the RD9 locus. A total of 125 MTC isolates were tested, as summarized in Table 1. Patterns differed by the presence or absence of the various PCR fragments in ways that were generally consistent per MTC species, but unexpected lineage- or strain-specific patterns sometimes resulted for certain isolates, as described in Table 1. PCR products and the 100-bp ladder (unlabeled lane) were visualized by agarose gel electrophoresis and ethidium bromide staining. Images were captured with the Nighthawk Imaging System (PDI Inc.) and Quality One software package (PDI Inc.). Lanes: 1, 16S rRNA gene; 2, cfp32 (Rv0577); 3, MiD3 (IS1561′); 4, RD4 (Rv1510); 5, RD7 (Rv1970); 6, RD1 (Rv3877-3878); 7, RD9 (Rv2073c); and 8, RD12 (Rv3120).

FIG. 2.

Summary diagram and phylogenetic interpretation of data collected in the current study. Shown are the various major divisions of the MTC segregated according to the presence or absence of the investigated inter-, intra-, and lineage-specific polymorphisms. Not included are most strain-specific SNPs and LSPs that were identified, as well as the potentially novel RD loci that were noted but not characterized in this study. Circles are placed at points in evolutionary history beyond which each strain that was evaluated possessed a consistent set of polymorphisms. The circles are numbered in the figure to denote the following: circle 1, RD12^can, hsp65⁶³¹, gyrB^can, mmpL6¹⁸⁷⁹, oxyR¹⁸³, pncA¹³⁸, Rv0911²⁴⁹, RD13²⁵⁵, and aroA⁹¹; 2, PPE55^can; 3, TbD1; 4, N-RD25^tbA; 5, RD1^tbB; 6, N-RD25^tbB; 7, pks15/1 (7-bp deletion) and katG⁴⁶³; 8, aroA¹¹⁷; 9, gyrA⁹⁵; 10, RD9 and gyrB^Δ (1450G→T); 11, RD713, TbD1¹⁹⁷, and aroA²⁸⁵; 12, RD711; 13, RD7, RD8, RD10, pks15/1 (6-bp deletion), 3′ cfp32³¹¹, RD13¹⁷⁴, PPE55²¹⁴⁸, and PPE55²¹⁵⁴; 14, Rv1510¹¹²⁹; 15, RD701, RD702, and hsp65⁵⁴⁰; 16, rpoB¹¹⁶³; 17, rpoB¹⁰⁴⁹; 18, RD1^das, N-RD25^das, 3′cfp32²²⁴, and Rv0911³⁸⁹; 19, mmpL6⁵⁵¹; 20, gyrB^oryx, TbD1¹⁷¹, PPE55²¹⁶², and PPE55²¹⁶³; 21, MiD3 and RD13³⁸⁰; 22, gyrB^mic, 16S rRNA¹²³⁴, and RD13⁶⁷; 23, 16S rRNA¹²⁴⁹ and RD13²²⁸; 24, RD12, RD13, N-RD25^bovis/cap, gyrB^Δ (756G→A), and oxyR²⁸⁵; 25, gyrB^Δ (1311T→G); 26, RD4, gyrB^Δ (1410C→T), and pncA¹⁶⁹; and 27, RD1^BCG. Note that distances are arbitrary and do not reflect the number of phylogenetically relevant polymorphisms present at each juncture. Although not evaluated in this study, as discussed in the text, data suggest that the −215 narGHJI SNP would also occupy circle 3. The loss of spoligotype spacers 33 to 36 may additionally correlate with circle 7, the absence of spacers 9 and 39 with circle 10, the deletion of spacers 40 to 43 with an event subsequent to divergence of the oryx bacillus putatively at circle X, and the deletion of spacers 3 and 16 with circle 24 of the above-proposed phylogeny (8, 40, 43, 55, 83).