Genomic variability of Mycobacterium tuberculosis strains of the Euro-American lineage based on large sequence deletions and 15-locus MIRU-VNTR polymorphism

Abstract

A sample of 260 Mycobacterium tuberculosis strains assigned to the Euro-American family was studied to identify phylogenetically informative genomic regions of difference (RD). Mutually exclusive deletions of regions RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 and RD761 were found in 202 strains; the RD(Rio) deletion was detected exclusively among the RD174-deleted strains. Although certain deletions were found more frequently in certain spoligotype families (i.e., deletion RD115 in T and LAM, RD174 in LAM, RD182 in Haarlem, RD219 in T and RD726 in the "Cameroon" family), the RD-defined sublineages did not specifically match with spoligotype-defined families, thus arguing against the use of spoligotyping for establishing exact phylogenetic relationships between strains. Notably, when tested for katG463/gyrA95 polymorphism, all the RD-defined sublineages belonged to Principal Genotypic Group (PGG) 2, except sublineage RD219 exclusively belonging to PGG3; the 58 Euro-American strains with no deletion were of either PGG2 or 3. A representative sample of 197 isolates was then analyzed by standard 15-locus MIRU-VNTR typing, a suitable approach to independently assess genetic relationships among the strains. Analysis of the MIRU-VNTR typing results by using a minimum spanning tree (MST) and a classical dendrogram showed groupings that were largely concordant with those obtained by RD-based analysis. Isolates of a given RD profile show, in addition to closely related MIRU-VNTR profiles, related spoligotype profiles that can serve as a basis for better spoligotype-based classification.

Figure 1. Minimum spanning tree based on MIRU-VNTR profiles of a set of 15 loci (424, 577, 580, 802, 960, 1644, 1955, 2163, 2165, 2401, 2996, 3192, 3690, 4052, and 4156) of M. tuberculosis clinical isolates bearing deletions RD115, RD122, RD 174, RD182, RD183, RD193, RD219 and RD726 (respectively indicated in different colours in the legend at the right side of the figure) and in isolates with no deletion (dark grey).

Each small-size circle represents a single isolate; larger circles represent clusters of 2 or 3 isolates, depending on the circle size, with identical MIRU-VNTR profiles; larger circles with more than one colour represent clusters including 2 or 3 isolates with identical MIRU-VNTR profiles but differing in RD deletions. The length of the lines is not proportional to the number of allelic variation between the isolates; an on-line supplemented file (Figure S1) is provided to visualize the allelic differences on the connecting lines.

Figure 2. Description of the five major RD-defined sublineages of the Euro-American lineage based on katG463/gyrA95, 15-locus MIRU-VNTR and spoligotype polymorphism.

For each RD-defined sublineage, a MST was constructed; the length of the lines is not proportional to the number of allelic variation between the isolates; an on-line supplemented file (Figure S2) is provided to visualize the allelic differences on the connecting lines. The RD deletion and the Principal Genotypic Group (PGG), defined by katG463/gyrA95 polymorphism, are reported at the left of each MST. Dark and light blue circles represent strains included in clonal complexes defined by a maximum of three locus differences; white circles represent strains outside the clonal complexes. Larger circles represent clusters of 2–3 isolates. At the right of each MST, the 15-locus MIRU-VNTR profile(s) of the central node strain(s) and the prevalent spoligotype patters (i.e, shared by at least 2 isolates) are provided; spoligotype patterns are shown in the conventional binary format; the spoligotype families, assigned to isolates on the basis of SITVITWEB database , are also shown at the right.