Clonality and micro-diversity of a nationwide spreading genotype of Mycobacterium tuberculosis in Japan

Abstract

Mycobacterium tuberculosis transmission routes can be estimated from genotypic analysis of clinical isolates from patients. In Japan, still a middle-incidence country of TB, a unique genotype strain designated as 'M-strain' has been isolated nationwide recently. To ascertain the history of the wide spread of the strain, 10 clinical isolates from different areas were subjected to genome-wide analysis based on deep sequencers. Results show that all isolates possessed common mutations to those of referential strains. The greatest number of accumulated single nucleotide variants (SNVs) from the oldest coalescence was 13 nucleotides, indicating high clonality of these isolates. When an SNV common to the isolates was used as a surrogate marker of the clone, authentic clonal isolates with variation in a reliable subset of variable number of tandem repeat (VNTR) genotyping method can be selected successfully from clinical isolates populations of M. tuberculosis. When the authentic clones can also be assigned to sub-clonal groups by SNVs derived from the genomic comparison, they are classifiable into three sub-clonal groups with a bias of geographical origins. Feedback from genomic analysis of clinical isolates of M. tuberculosis to genotypic markers will be an efficient strategy for the big data in various settings for public health actions against TB.

Fig 1. Profiles of 10 M-strain isolates subjected to genomic comparison.

Locations of the origins are pointed on a geographical map of Japan.

Fig 2. Phylogenetic tree of 10 clonal isolates of M-strain based on SNVs identified from genomic comparison.

Two referential strains, T85 and CCDC5079, are also depicted according to the SNVs to H37Rv. The isolates were assigned topologically to three sub-clonal groups (Group I, KK11-2645, KK11-2647, 4991, 080715T, and 090824T; Group II, Oki-1530 and Yg10-015; and Group III, FY21N052, 5984S and 6503R). Positions of SNVs on whole genome sequence of H37Rv (AL123456.2) were put on each branch.

Fig 3. Numbers of SNVs between pairwise isolates of 10 M-strains identified from genomic comparison.

Fig 4. Phylogenetic positions of 53 clonal isolates of M-strains identified using a surrogate SNV marker (position 1757565, G to A).

The tree is topologically identical to Fig. 2. Circles on the tree denote clones isolated from respective areas (Yamagata, Tokyo, Osaka, Kobe, and Okinawa). Sizes and numbers of circles show the number of clones. Colours in the circles represent years of isolation.